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utions Cell

The Newsletter for Cell Biology Researchers

Vol 2: 2011

Change Cell Fate


Enhanced Reprogramming of Human Somatic Cells Using Human STEMCCA Polycistronic Lentivirus and Human iPS Cell Boost Supplement page 3
Fluorescent Gelatin Degradation Assays for Investigating Invadopodia Formation Page 8 Exploring Akt/mTOR Signaling Using the MILLIPLEX map Akt/mTOR 11-plex Panel Page 13 Immuno-monitoring Using the Scepter 2.0 Cell Counter and Software Module Page 16 Mitochondrial to Nuclear DNA Ratio: Sensitive Biomarker of Stem Cell Differentiation Page 23 Exciting New Products for Cell Biology Research Page 27

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Enhanced Reprogramming of Human Somatic Cells using Human STEMCCA Polycistronic Lentivirus and Human iPS Cell Boost Supplement
Min Lu, Cristina Moore, Vi Chu EMD Millipore

Abstract
The ability to reprogram differentiated adult cells to a state that resembles embryonic stem cells has created wide-ranging opportunities for development of relevant in vitro disease models and patient-specific cell replenishment therapies. Initial efforts to generate human induced pluripotent stem cells (iPS cells) required simultaneous co-infection of cells with four separate retroviral expression vectors (Oct-4, Klf4, Sox-2 and c-Myc). Each vector carried one transcription factor, which resulted in a high number of genomic integrations. Single polycistronic lentiviral vectors, such as those in EMD Millipores STEMCCA lentivirus reprogramming kits, can improve efficiency and reduce the number of viral integrations. In one report, high percentages of disease-specific human iPS clones were isolated which possessed only a single viral integrant .
2

fibroblasts (MEFs) and human foreskin fibroblasts (HFFs), respectively. However, a high multiplicity of infection (MOI = 200) was required for reprogramming human somatic cells. The recently launched human STEMCCA kits replaced mouse genes with human transcription factors, yielding similar numbers of colonies but at a dramatically reduced MOI (MOI = 20). Both human and mouse STEMCCA lentivirus kits are available in constitutive and Cre/LoxPregulated formats for higher reprogramming efficiency of normal and diseased post-natal somatic cells2-4. Although lenti- and retro-virus based reprogramming remain the most efficient methods to deliver exogenous reprogramming factors into the host cell genome, human iPS cell generation is still slow (around 4 weeks) and inefficient (0.01-0.1% efficiency), resulting in mixed populations of partial and full reprogrammed colonies. Substantial effort and progress have been made to generate iPSCs with fewer or no exogenous genetic manipulations for example, by introduction of chemical compounds that can functionally replace reprogramming transcription factors and/or enhance efficiency and kinetics of reprogramming5-7. Human keratinocytes8 and mouse fibroblasts9 have now been reprogrammed to iPSCs with a single gene, Oct4, and a cocktail of small molecules. Here, various combinations of small molecules involved in TGFa, Wnt, and MAPK signaling pathways along with epigenetic modifiers were screened for their effects on (1) increasing the ratio of fully reprogrammed SSEA4+TRA-1-60+ Hoechstdim iPS cells versus reprogramming intermediates; (2) increasing colony numbers and (3) reducing the time to establishment of full reprogrammed iPS cell colonies.

Despite these advances, reprogramming human somatic cells remains a highly inefficient and time-consuming process. Small molecules targeting specific signaling pathways have been shown to enhance reprogramming and/or replace the transcription factors required for reprogramming5-7. Here, a cocktail of small molecules was identified that, when used in conjunction with the STEMCCA kits, further increased reprogramming efficiency, decreased time required to establish full reprogramming, and maintained the desired iPS cell morphology and pluripotency.

Introduction
We have previously developed mouse STEMCCA lentivirus kits that have been validated for the generation of both mouse and human iPS cells from mouse embryonic

Seed HFFs or target cells Infect with virus Infect with virus
0 1 2 3 4

Prepare feeders Replate on feeders Add feeders to culture


5 6 7 8 9 10 11 12 13 14 15 16

Prepare feeders Pick colonies

Day Medium

17

18

19

20

21

22

23

24

25

FibroGRO LS Complete Medium MEF

Human ESC Media MEF Human iPS Cell Boost Supplement

Figure 1. Time course schematic of reprogramming human somatic cells using STEMCCA polycistronic lentivirus kits combined with Human iPS Cell Boost Supplement.

From this screen, a small molecule boost supplement was identified which in combination with the STEMCCA lentivirus kits fulfilled the three aforementioned criteria. The small molecule boost supplement was validated on feeder-based (KOSR) and serum-free, feeder-free culture systems and was furthermore shown to be highly effective in reprogramming different primary fibroblast cell lines.

treatments were able to give rise to large flat 2D colonies that displayed morphological features similar to hESCs and could be easily passaged. Colonies were stained with anti-Tra-1-60 (Cat. No. FCMAB115F) and anti-SSEA-4 (Cat. No. MAB4304). Tra-1-60 is a marker that is expressed at a later stage of reprogramming10. Tra-1-60 and SSEA-4 double positive staining has been suggested to be a valid marker for full reprogrammed iPSCs11.

Materials and Methods


The reprogramming protocol followed is outlined in Figure 1. FibroGRO Xeno-free Human Foreskin Fibroblasts (HFFs, Cat No. SCC058) were seeded at a density of 10,000 cells/well and transduced with the Human STEMCCA Constitutive Polycistronic Lentivirus Reprogramming Kit (Cat No. SCR544). Cells were replated onto irradiated MEFs in the presence or absence of a panel of small molecules at day 6, using the human ESC medium of choice (either KOSR-based media on inactive MEFs (Cat No. PMEF-CF) or on Matrigel-coated or Geltrex- coated plates if using mTeSR or StemPRO media, respectively). The medium was changed every other day and colonies were picked when they reached several hundred cells in size. A total of 25 small molecule boost cocktails (proprietary formulations) were tested and several

results
HFF were transduced with STEMCCA lentivirus, replated onto irradiated MEFs in the presence or absence of a panel of small molecules at day 6, and evaluated for number of colonies generated, morphology, ease of passaging, and days to passage (Figure 2, 3). Chemically treated human iPSCs possessed fast proliferation kinetics; early passages from P0 to P3 were shortened to 5-6 days per passage period - a timeframe that is similar to the proliferation rate of normal hESC (Figure 3). Treatment 2 was selected on the basis that it significantly improved both the quality of colonies formed and the efficiency of reprogramming. Treatment 2 is herein referred to as Human iPS Cell Boost.

untreated Morphology: # Colonies: Passageability: Days to Passage: 3D 1x Difficult 10-12 days

treatment 1 Flat 2D 3.6x Easy 5-6 days

treatment 2 Flat 2D 2.6x Easy 5-6 days

treatment 3 Flat 2D 0.5x Easy 5-6 days

Figure 2. Small molecule combinations were screened for their effects on colony morphology, number of colonies generated, ease of passaging and relative proliferation rate as measured by days to passaging. Treatment 2 was selected for further characterization. Treatment 2 is referred to as Human iPS Cell Boost Supplement. 4

In the presence of the Human iPS Cell Boost Supplement, the number of colonies formed increased 3-fold (Figure 4A) when used in combination with the mouse STEMCCA lentivirus kits (Cat. Nos. SCR530, SCR531) and 15-fold when used in combination with the human STEMCCA lentivirus kits (Cat. Nos. SCR544, SCR545). Colonies stained positive for both human ESC markers, SSEA-4 (Figure 4F) and TRA-1-60 (Figure 4G). TRA-1-60+ colonies were not

treatment 1

treatment 2

treatment 3

P1 (5-6) P2 (5-6)

observed in the untreated control cultures (Figure 4D). Human iPS colonies generated in the presence of Human iPS Cell Boost Supplement could be readily expanded for multiple passages (over 30 passages). Human iPS cells displayed the morphology characteristic of human ESCs, had a normal karyotype and stained positive for pluripotent markers (Figure 5).
Figure 3. Proliferation kinetics similar to human ESC were obtained for all three chemical treatments starting at p1 (5-7 days till passaging) versus from p3 for untreated control (data not shown).

No. of colonies

25 20 15 10 5 0

Mouse STEMCCA

Cell Boost Treated

Control

70 60 50 40 30 20 10 0

Human STEMCCA

No. of colonies

Cell Boost Treated

Control

Figure 4. Addition of Human iPS Cell Boost Supplement to a polycistronic lentivirus-based reprogramming regime (STEMCCA) dramatically increased the efficiency of colony formation (A) and shortened the time to establishment of full reprogrammed human iPS clones (E, F, G). Human iPS Cell Boost Supplement enhanced colony formation by 2-3 fold when used in combination with the mouse STEMCCA lentivirus kits (SCR530, SCR531) and 15-fold when used in combination with the human STEMCCA lentivirus kits (SCR544, SCR545) (A). Four independent experiments (MOI = 200) were performed using mouse STEMCCA lentivirus kit while two experiments were performed (MOI = 10) using human STEMCCA lentivirus kits. Reprogramming without chemical treatment was used as a control for all experiments. p0 human iPS colonies generated from FibroGRO Xeno-free Human Foreskin Fibroblasts (Cat. No. SCC058) reprogrammed with mouse STEMCCA lentivirus (SCR530) in the presence of the Human iPS Cell Boost Supplement exhibited larger colony sizes, a flat 2D morphology (E), and are SSEA-4- positive (F) and TRA-1-60-positive (G). This is in marked contrast to untreated control where the colonies are smaller, are 3D in morphology (B) and are SSEA-4 positive (C) but TRA-1-60-negative (D) at similar timepoints.

Figure 5. Human iPS cells generated using the Human STEMCCA Cre-Excisable Constitutive Polycistronic (OKSM) Lentivirus Kit (SCR545) in combination with Human iPS Cell Boost Supplement possessed an apparently normal karyotype (A) and expressed the appropriate human pluripotent markers, alkaline phosphatase (B), SSEA-4 (C), Oct-4 (D), SSEA-3 (E), TRA-1-60 (F), and TRA-1-81 (G). Cytogenic analysis was performed on twenty G-banded metaphase cells from p9 human iPS cells. All twenty cells demonstrated an apparently normal male karyotype. No abnormal cells were detected (A, Cell Line Genetics). 5

The versatility of the Human iPS Cell Boost Supplement was further demonstrated by enhanced reprogramming of multiple primary human fibroblast cell lines including HFF and BJ (ATCC) (Figure 7) in either KOSR feeder-based or serum-free, feeder-free culture systems (mTeSR and StemPRO) (Figure 6).

C
untreated Control Cell Boost treated

E C D
No. of colonies
50 40 30 20 10

BJ Human Foreskin Fibroblast

Feeder/serum free culture mTeSR

No. of cells/well re-plated in feeder-free culture 5 x 104 105

Chemical treatment + + + + -

Cell Boost Treated

Control

Colony# 70 6 76 22 23 9 15 1 Figure 7. Day 12 human iPS colonies generated from BJ neonatal foreskin fibroblasts using mouse STEMCCA lentivirus kit in combination with Human iPS Cell Boost Supplement exhibited a 10-fold increase in colony numbers (E) and displayed increased colony size (B) relative to untreated control (A). Human iPS colonies picked at day 21 and passaged on MEFs exhibited similar flat 2D morphology (C, D) and similar proliferation kinetics as human ES cells (data not shown).

StemPro

5 x 104 10
5

Figure 6. Human iPS Cell Boost Supplement improves reprogramming efficiency in serum-free, feeder-free based culture systems and increased colony size relative to untreated controls (compare B, D to A, C). Human iPS colonies were generated using mouse STEMCCA lentivirus kit in the absence or presence of the Human iPS Cell Boost Supplement. Cells were cultured on either Geltrex-coated plates in StemPRO medium (A, B) or Matrigel-coated plates in mTeSR medium (C, D). Day 23 fully reprogrammed hiPS colonies were isolated and continually passaged in mTeSR conditions exhibited typical hESC morphology (F).

Conclusion
In summary, we have established a robust reprogramming method that is amenable to efficient reprogramming of different fibroblasts readily available from human donors. By using small molecules that modulate key signaling pathways and epigenetic modifiers, we could dramatically improve the quality and quantity of human iPS colonies generated. This novel method increased the number of human iPS colonies by 2-3 fold when the mouse STEMCCA lentivirus kit was used and up to 15-fold when the human STEMCCA lentivirus kit was used. The colonies formed possessed the distinctive flat 2D morphology that are more reminiscent of human embryonic stem cells and could be easily passaged in contrast to untreated control that exhibited 3D morphology and were more difficult to isolate. Furthermore, the time to establishment of fully reprogrammed human iPS colonies that are SSEA4+TRA1-60+Hoechstdim was significantly shortened by 50%. Reprogramming with the small molecule boost supplement was further validated on multiple human fibroblast cell lines in feeder-free and serum-free culture systems. In summary, the use of the STEMCCA polycistronic lentivirus in combination with the small molecule boost supplement provides a convenient solution for enhanced reprogramming efficiency.

references 1. Sommer CA, et al. (2009) iPS cell generation using a single lentiviral stem cell cassette. Stem Cells 27: 543-549. 2. Somers A, et al. (2010) Generation of transgene-free lung disease-specific human iPS cells using a single excisable lentiviral stem cell cassette. Stem Cells 28: 1728-1740. 3. Tchieu J, et al. (2010) Female human iPSCs retain an inactive X chromosome. Cell Stem Cell 7: 329-342. 4. Buecker C, et al. (2010) A murine ESC-like state facilitates transgenesis and homologous recombination in human pluripotent stem cells. Cell Stem Cell 6: 535-546. 5. Huangfu D, et al. (2008) Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nat. Biotechnol. 26: 1269-1275. 6. Ichida JK, et al. (2009) A small-molecule inhibitor of TGF-Beta signaling replaces sox2 in reprogramming by inducing nanog. Cell Stem Cell 5: 491-503. 7. Lin T, et al. (2009) A chemical platform for improved induction of human iPSCs. Nat. Methods 6: 805-808. 8. Zhu S, et al. (2010) Reprogramming of human primary somatic cells by Oct4 and chemical compounds. Cell Stem Cell. 7:651-655. 9. Li Y, et al. (2010) Generation of iPSCs from mouse fibroblasts with a single gene, Oct4, and small molecules. Cell Research. 1-9. 10. Chan E, et al. (2009) Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat. Biotechnol. 27:1033-1037. 11. Hockemeyer D, et al. (2008) A drug-inducible system for direct reprogramming of human somatic cells to pluripotency. Cell Stem Cell. 3:346-353.

RELATED PRODUCTS
Available from www.millipore.com.
Description Catalogue No.

Human iPS Cell Boost Supplement Human STEMCCA Constitutive Polycistronic (OKSM) Lentivirus Reprogramming Kit Human STEMCCA Cre-Excisable Constitutive Polycistronic (OKSM) Lentivirus Reprogramming Kit FibroGRO Xeno-Free Human Foreskin Fibroblasts FibroGRO LS Complete Medium

SCM088 SCR544 SCR545 SCC058 SCMF002

Fluorescent Gelatin Degradation Assays for Investigating Invadopodia Formation


Janet Anderl, Jun Ma and Luke Armstrong EMD Millipore

Abstract
The invasion of cells through tissue and associated extracellular matrix is a critical activity in both physiological and pathological processes, such as embryological development and cancer metastasis. A key cellular feature involved in matrix degradation is the formation of protrusions of localized protease activity, termed invadopodia or podosomes. An effective method for visualizing subcellular invadopodia formation involves the plating of cells onto a thin layer of fluorescentlylabeled matrix. Areas of degradation are associated with a loss of fluorescence, and these regions may be microscopically imaged and colocalized with molecules of interest in the proteolytic pathway. Here we demonstrate the capabilities of two new invadopodia assay kits, which provide the reagents necessary for generating thin coatings of prelabeled fluorescent gelatin on glass substrates. These kits enable simple, rapid, and consistent production of homogeneous gelatin matrices and visualization of degradation produced by multiple cell types. Furthermore, degradation may be quantified by image analysis and used to characterize proteolytic time-courses and modulator effects on invadopodia formation.

for analyzing invasion at the cell population level, but analysis of the subcellular events mediating the stages of invasion requires techniques with higher resolution. The method that has been most informative for pinpointing regions of the cell that initiate invasion involves plating cells on a culture surface coated with a thin layer of fluorescently-labeled matrix, and visualizing regions where the cell has degraded the matrix to create an area devoid of fluorescence2. Such assays have revealed that invasive cells extend small, localized protrusions that preferentially degrade the matrix. These protrusions are termed invadopodia in cancerous cells, and podosomes in non-malignant cells such as macrophages3. Many molecules orchestrate the formation and function of invadopodia; a few of the key molecular events include Src phosphorylation of scaffolding protein Tks54, N-WASP activation and cortactin regulation of the Arp2/3 complex to induce actin polymerization5,6, generation of reactive oxygen species by NADPH oxidases7, and cortactin-mediated localization of membrane-type and secreted matrix metalloproteases (MMPs) to the invadopodia8. EMD Millipores QCM Gelatin Invadopodia Assays provide optimized materials and protocols to enable reproducible analysis of invadopodia in invasive tumor cells (Catalog No. ECM670 for green fluorescence, Catalogue No. ECM671 for red fluorescence). Reagents are provided for coating glass culture surfaces with fluorescent matrix and for colocalizing the actin cytoskeleton and nuclei with invadopodial degradation sites. This assay may also be used for assessing the activity of inhibitors and promoters of invadopodia formation and function. Furthermore, different cell types and individual cells in heterogeneous populations may be analyzed for invasive potential. Finally, the assay kits provide troubleshooting suggestions, recommendations for coating on multiple substrate formats, and example studies in several assay systems (e.g., various cell types, time-course studies, degradation modulation).

Introduction
Invasion of cells through layers of extracellular matrix is a key step in tumor metastasis, inflammation, and development. Stages of invasion include adhesion to the matrix, degradation of proximal matrix molecules, extension and traction of the cell on the newly revealed matrix, and movement of the cell body through the resulting gap in the matrix . Each of these stages is executed by a suite of
1

proteins, including proteases, integrins, GTPases, kinases, and cytoskeleton-interacting proteins. Classical methods for analyzing cellular invasion involve application of cells to one side of a layer of gelled matrix molecules and quantifying the relative number of cells that have traversed the layer. Such methods are extremely useful
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Assay Principle
The EMD Millipore QCM Gelatin Invadopodia Assays provide the reagents necessary for affixing a thin, uniform layer of pre-labeled fluorescein (green)- or Cy3 (red)gelatin to a glass culture substrate, allowing for rapid detection of matrix degradation9,10. A poly-L-lysine coating is first adsorbed to the glass substratum. The substrate is then treated with a dilute glutaraldehyde solution to bifunctionally activate the surface for further protein binding. Subsequent incubation of the surface with fluorescent gelatin allows covalent coupling between the poly-L-lysine and gelatin via reactive aldehyde (-CHO) groups. The fluorescently-coated glass is now prepared for cell culture by disinfection with 70% ethanol, followed by quenching of free aldehydes with amino acid-containing growth medium. Upon completion of fluorescent substrate preparation, cell types of interest may be seeded onto the gelatin surface for a desired amount of time. Depending on cell type, degradation may occur within a few to several hours, and treatment compounds of interest may also be introduced within the culture period (Figure 1). Degraded areas of gelatin, now devoid of fluorescence, may be microscopically visualized and quantified using image analysis software. The assay also provides fluorescentlylabeled phalloidin (TRITC- or FITC-conjugated) and DAPI, for visualization of cytoskeletal F-actin and nuclei, respectively, to allow colocalization of degradation with cellular features. Potential activators or inhibitors of invadopodia formation may be investigated for their influence on the degree and frequency of matrix degradation, and the assay may be further combined with immunocytochemical staining for other molecules of interest in mechanistic studies.

poly-L-lysine in deionized water for 20 minutes at room temperature (RT). The poly-L-lysine was then removed, and the slides rinsed three times with 500 L/well of Dulbeccos PBS (DPBS). Next, 250 L of dilute glutaraldehyde in DPBS was added to each well for 15 minutes at RT to activate the poly-L-lysine surface for further protein attachment. Following removal of the glutaraldehyde, each well was again rinsed three times with 500 L of DPBS. Finally, 200 L of gelatin in DPBS, mixed at a 1:5 ratio of fluorescentlylabeled:unlabeled gelatin, was coated onto each well for 10 minutes at RT, followed by three rinses in DPBS. All steps including, and subsequent to, fluorescent-gelatin coating were performed so as to protect the glass slides from photobleaching due to excessive exposure to light. To prepare for cell plating, the gelatin substrates were disinfected with 500 L/well of 70% ethanol for 30 minutes at RT. After ethanol removal and rinsing in DPBS, free aldehydes were quenched by the addition of 500 L/well of amino-acid-containing growth medium and incubated at RT for 30 minutes. Cell types of interest were detached and seeded as described in the previous section. For some experiments, a modulator of invadopodia formation, focal adhesion kinase inhibitor II (5 M final concentration, or 0.4% DMSO control), was added simultaneously with plating. Sample Fixation and Staining At the desired time-point after plating, growth medium was removed from the chamber slides and the samples were fixed for 30 minutes at RT with 250 L/well of 3.7% formaldehyde in DPBS. Samples were then rinsed twice with 500 L/well of fluorescent staining buffer (DPBS with 2% blocking serum and 0.25% Triton X-100 for cell
6. Cell type/treatment of Interest in Growth Medium (e.g 24 hr, 37 C) 5. Growth Medium (30 min RT) 4. 70% Ethanol (30 min RT)

Materials and Methods


Cell Lines used MDA-MB-231 human breast adenocarcinoma, RPMI-7951 and SK-MEL-28 human skin melanoma, and IC-21 mouse peritoneal macrophages were obtained from ATCC and cultured to 80-90% confluence in tissue culture flasks. For seeding onto gelatin surfaces, cells were detached using 0.25% trypsin-EDTA (or DPBS without calcium and magnesium for IC-21 cells), pelleted, then resuspended in growth medium to a concentration of 28,000 cells/mL (20,000 cells/cm2). Cells were seeded in a volume of 500 L/well and cultured for the desired duration of degradation, generally between 8-48 hours. Substrate Preparation and Cell Seeding To facilitate attachment of fluorescent gelatin, 8-well glass chamber slides were first coated with 250 L/well of dilute
3. Fluorescent Gelatin (10 min RT) 2. Glutaraldehyde (15 min RT) 1. Poly-L-Lysine (20 min RT)

Figure 1. Gelatin invadopodia assay setup.

CHO CHO CHO CHO CHO CHO CHO CHO CHO CHO CHO CHO CHO CHO
Glass Substrate

CHO CHO CHO CHO CHO CHO CHO CHO CHO CHO CHO CHO CHO CHO

DAPI Nuclear Count

Phalloidin total Cell Area

Fluorescent Gelatin Degradation Area

Imaging and Analysis Mounted cover glasses were allowed to hard-set before fluorescent imaging with illumination and filters appropriate for fluorescein/FITC, Cy3/TRITC and DAPI excitation and emission wavelengths. Samples were imaged on an inverted wide-field fluorescent microscope at 20X objective magnification for quantification studies (5 fields of view per well) or at 63X objective magnification (oil

threshold

threshold and Area Measurement

threshold and Area Measurement

immersion) for colocalization experiments. Image analysis was performed using free, downloadable ImageJ software distributed by the National Institutes of Health (NIH)10. A high intensity threshold was set for positive DAPI signal, then analyzed as particles for determination of a nuclear (cell) count. Similarly, setting a high intensity threshold for phalloidin signal enabled

Count

measurement of total cell area per field of view. Conversely, permeabilization). For immunocolocalization studies, 200 L of primary antibody in fluorescent staining buffer was added to each well for 1 hour incubation at RT. Samples were then rinsed three times with 500 L/well of fluorescent staining buffer before proceeding on to 1 hour RT incubation with a low intensity threshold was set for diminished fluorescent gelatin signal to enable quantification of total degradation area per field of view (Figure 2).

Figure 2. Example image analysis with NIH ImageJ software.

fluorescent secondary antibody, fluorescently-conjugated phalloidin (2 g/mL) and DAPI (1 g/mL) in staining buffer. Primary and secondary antibodies were omitted for stains incorporating phalloidin and DAPI only. Finally, samples were rinsed twice each with fluorescent staining buffer and DPBS before removal of culture chambers and cover-slipping. Slide-mounting medium contained anti-fade reagent and appropriately thick cover glasses were selected for imaging magnification of choice.

results and Discussion


We demonstrated the ability to visualize and quantify the degradation of fluorescent gelatin matrices by a variety of cell types. For the cell lines MDA-MB-231, RPMI-7951, and IC-21, gelatin proteolysis demonstrated a range of degradation patterns that may be attributed to invadopodia or podosome formation, including punctate, linear, or blotchy areas devoid of fluorescein-gelatin fluorescence (Figure 3). Often, not all cells in a population will exhibit proteolytic behavior, and cellular movement between sites of degradation may frequently be observed. SK-MEL-28 cells, a noninvasive melanoma type, did not display gelatin degradation (Figure 3).

MDA-MB-231 (Human Breast Adenocarcinoma)

rPMI-7951 (Human Skin Melanoma)

SK-MEL-28 (Human Skin Melanoma)

IC-21 (Mouse Peritoneal Macrophage)

Fluorescein-Gelatin

trItC-Phalloidin/DAPI

Figure 3. Fluorescent gelatin degradation and phalloidin/DAPI staining of multiple cell types. Fluorescein-gelatin matrices (top panel, green) were coated onto 8-well glass chamber slides. Multiple human cancer cell lines (MDA-MB-231, RPMI-7951, and SKMEL-28) and a mouse macrophage cell line (IC-21) were plated onto the gelatin substrates at 20,000 cells/cm2 for a culture duration of 24 hours. F-actin and nuclei were stained, respectively, with TRITC-phalloidin (bottom panel, red) and DAPI (bottom panel, blue). Cells were imaged at 63X objective magnification (bar = 25 m). 10

Colocalization Studies In Figure 4, RPMI-7951 cells seeded onto Cy3-gelatin matrices demonstrated the ability to co-localize sites of gelatin degradation with phalloidin (F-actin) puncta and cortactin foci. Cortactin protein is strongly associated with actin assembly, and colocalization of this molecule with areas of proteolysis was indicative of dynamically active invadopodia formation (see white arrows in figure 4). time-dependent Degradation Over 100 cells per condition were analyzed to obtain the percent degradation area of total cell area data depicted in Figure 5. For MDA-MB-231 and IC-21 cells, degradation percentage increased over time (particularly between 8 and 24 hour time points), whereas no degradation by noninvasive SK-MEL-28 cells was observed. Of note is that although the theoretical maximum of percent degradation area of total cell area is 100%, higher amounts of degradation were observed here, likely due to cellular
% Degraded Area

DAPI

Cy3-Gelatin

FItC-Phalloidin

Cy5-Cortactin Antibody

Gelatin/ Phalloidin Overlay

Figure 4. Colocalization of degradation with invadopodia-related puncta. Cy3-gelatin matrices were coated onto glass chamber slides, and RPMI-7951 human skin melanoma cells were seeded onto the gelatin substrates for 24 hours. For fluorescent immunocytochemistry, cells were incubated with anti-cortactin, followed by detection with a Cy5conjugated secondary antibody. Secondary antibody incubation was performed concurrently with FITC-phalloidin and DAPI staining. Cells were imaged at 63X objective magnification.

Cy3-Gelatin: % Degradation Area of Total Cell Area


125 100 75 50 25 0 MDA-MB-231 SK-MEL-28 Cell Type IC-21 8 hr 24 hr 48 hr

movement during proteolysis. Such historical degradation is recorded using this assay, resulting in degradation areas larger than the area of a cell itself (particularly for longer time points or highly motile cell types). Modulation of Matrix Degradation Cells were seeded onto fluorescein-gelatin matrices and simultaneously treated with focal adhesion kinase (FAK) inhibitor II (PF-573,228) or a DMSO control (Figure 6). FAK inhibition, which has previously been shown to enhance invadopodia formation in certain cell types12, was indeed observed to increase MDA-MB-231-associated degradation over the course of 24-hour treatment. The non-invasive phenotype of SK-MEL-28 cells was not altered by addition of FAK inhibitor II, but surprisingly, degradation by IC-21 cells was decreased by treatment with the compound. Such opposite effects as those seen between the MDA-MB-231 and IC-21 cells emphasize variations in proteolytic behavior between cell types, and may be indicative of significant differences in degradation signaling mechanisms between cancerous and normal cell phenotypes.

8 hr

24 hr Cy3-Gelatin

48 hr

FItC-Phalloidin/DAPI

Figure 5. time-dependent gelatin degradation. Multiple cell types (images are of MDA-MB-231) were plated onto Cy3-gelatin substrates (top image panel, red) and cultured for 8, 24, or 48 hours. Following staining with FITC-phalloidin (bottom image panel, green) and DAPI (bottom image panel, blue) cells were imaged at 20X objective magnification at 5 fields of view per well. Bar = 100 m. Percent degradation area of total cell area was quantified using ImageJ analysis software, as depicted in Figure 2.

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Conclusion
Our results demonstrate the utility of EMD Millipores QCM Gelatin Invadopodia Assays in the visualization and quantification of gelatin degradation by a variety of cell types at multiple time points and following treatment with modulators of invadopodia formation. Specifically, we show that both time-dependence of matrix degradation and efficacy of invadopodia modulators are heavily influenced by cell type, inviting further, detailed studies of the differential signaling controlling these processes. To this end, QCM Gelatin Invadopodia Assays can provide a convenient, flexible system for monitoring matrix degradation and investigating key players in the proteolytic process, at the single cell and subcellular levels.

Fluorescein-Gelatin: % Degradation Area of Total Cell Area


% Degraded Area 40 30 20 10 0 MDA-MB-231 SK-MEL-28 Cell Type IC-21 DMSO Control 5 M FAK Inhibitor II

references 1. Friedl P and Wolf K. Plasticity of cell migration: a multiscale tuning model. J Cell Biol 2010; 188:11-19. 2. Chen WT et al. Expression of transformation-associated protease(s) that degrade fibronectin at cell contact sites. J Cell Biol 1984; 98:1546-1555. 3. Ayala I et al. Invadopdia: a guided tour. Eur J Cell Biol 2006; 85:159-164. 4. Seals DF et al. The adaptor protein Tks5/Fish is required for podosome formation and function, and for the proteasedriven invasion of cancer cells. Cancer Cell. 2005; 7:155-165. 5. Yamaguchi H et al . Molecular mechanisms of invadopodium formation: the role of the N-WASP-Arp2/3 complex pathway and cofilin. J Cell Biol 2005; 168: 441452. 6. Weaver AM. Invadopodia: specialized cell structures for cancer invasion. Clin Exp Metastasis. 2006; 23:97-105. 7. Diaz B et al. Tks5-dependent, Nox-mediated generation of reactive oxygen species is necessary for invadopodia formation. Sci Signal 2009; 2:ra53. 8. Clark ES and Weaver AM. A new role for cortactin in invadopodia: regulation of protease secretion. Eur. J. Cell Biol 2008; 87: 581-590. 9. Artym VV, Yamada KM and Mueller SC. ECM degradation assays for analyzing local cell invasion. Methods Mol Biol 2009; 522:211-219. 10. Xu X, Johnson P and Mueller SC. Breast cancer cell movement: imaging invadopodia by TIRF and IRM microscopy. Methods Mol Biol. 2009; 571: 209-225. 11. Rasband WS (1997-2011), ImageJ, US National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij/. 12. Liu S. et al. Laminin-332-b1 integrin interactions negatively regulate invadopodia. J Cell Physiol. 2010; 223:134-142.

MDA-MB-231 (DMSO Control)

MDA-MB-231 (5 M FAK Inhibitor II)

IC-21 (DMSO Control)

IC-21 (5 M FAK Inhibitor II)

Fluorescein-Gelatin

trItC-Phalloidin/DAPI

Figure 6. Modulation of gelatin degradation by a FAK inhibitor. Fluorescein-gelatin matrices (top image panel, green) were seeded with MDA-MB-231, SK-MEL-28, or IC-21 cells and simultaneously treated with 5 M FAK inhibitor II or a 0.4% DMSO control. Following 24 hour treatment, cells were fixed and stained for F-actin and nuclei with TRITC-phalloidin (bottom image panel, red) and DAPI (bottom image panel, blue). Samples were imaged at 20X objective magnification at 5 fields of view per well. Bar = 100 m.

ORDER INFO & RELATED PRODUCTS


Available from www.millipore.com.
Description Catalogue No. Description Catalogue No.

QCM Gelatin Invadopodia Assay (Green) QCM Gelatin Invadopodia Assay (Red) Mouse Anti-Cortactin (p80/85), Clone 4F11 Mouse Anti-MMP-14 [MT1-MMP], Clone LEM-2/15.8 Mouse Anti-Src, Clone GD11 Rabbit Anti-Tks5 (SH3 #1) Rabbit Anti-N-WASP Rabbit Anti-Arp2 12

ECM670 ECM671 05-180 MAB3328 05-184 09-403 AB2962 AB3886

Rabbit Anti-Arp3 GM6001 [Ilomastat] MMP Inhibitor 0.25% Trypsin-EDTA in Hanks Balanced Salt Solution EmbryoMax 1X Dulbeccos Phosphate Buffered Saline Available from www.emdbiosciences.com. Focal Adhesion Kinase Inhibitor II PP2 (Src Inhibitor)

07-272 CC1100 SM-2003-C BSS-1006-B

324878 529573

Exploring Akt/mTOR Signaling Using the MILLIPLEX map Akt/ mTOR 11-plex Panel
Joseph Hwang, Ph.D. EMD Millipore

Introduction
The Akt signaling pathway, one of the most oftendysregulated signaling pathways in cancer, plays an important role in mediating a very broad range of cellular processes such as growth and development, cell cycle, energy homeostasis and survival. Akt, a serine/threonine kinase that phosphorylates over 100 protein substrates, mediating cell survival and proliferation signals, is often itself hyperphosphorylated in various tumor types. Although the Akt gene itself is not known to be mutated in cancers, many of the upstream regulators of Akt, including IR, IRS, PI3K and PTEN, are oncogenes and tumor suppressors. Downstream of Akt, the mammalian Target Of Rapamycin (mTOR) complex is a key regulator of growth and metabolism. As nearly all of the players in the Akt/mTOR signaling pathway are coordinately regulated by phosphorylation, understanding the role of this pathway in normal physiological processes and in diseases such as cancer and diabetes requires the ability to simultaneously measure phosphorylation status of multiple protein targets. Several assays to examine phosphorylation status are currently available, including Western blotting, ELISA, reverse phase arrays, quantitative cell imaging, and mass spectroscopy. Although some of these platforms yield absolute, quantitative data, the assays are either limited to measuring only one analyte at a time, or are excessively difficult or expensive. On the level of multiparametric single-cell analysis, flow cytometry has enabled the study of multiple pathway activation and cross-talk in a time-dependent manner. Directly applicable to the Akt/mTOR pathway, EMD Millipores FlowCellect PI3K-mTOR Assay Kit uses directly conjugated antibodies against phospho-Akt1/PKB (Ser473) and phospho-ribosomal protein S6 (Ser235) to analyze both upstream and downstream portions of this signaling cascade.

Bead-based assays, such as those using Luminex xMAP technology, have enabled the measurement of phosphorylation levels of up to 12 proteins simultaneously. EMD Millipores MILLIPLEX map Akt/mTOR Panel is a bead-based immunoassay that simultaneously detects 11 phosphoproteins (Figure 1) in the Akt/mTOR pathway in a single cell lysate sample, enabling the measurement of phosphorylation changes in this important pathway. Here, we demonstrate the utility of the MILLIPLEX map Akt/mTOR 11-plex Panel in the analysis of Akt/mTOR signaling in cancer cell lines (HepG2, HEK293, and MCF7). All analytes were detected with good specificity, sensitivity and precision. In addition, we demonstrate successful detection of these phosphoproteins in human as well as mouse tissue samples (with the exception of phosphoIGF1R in mouse samples). Finally, our inhibitor studies show the utility of this panel in drug discovery research.

PDK1

PTE

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Figure 1. the Akt/mtOr signaling pathway. Analytes included in the MILLIPLEX map Akt/mTOR 11-plex Panel are highlighted in red. 13

Methods
tissue Culture HepG2, HEK293, and MCF7 cells were cultured according to ATCC guidelines in recommended media. Cells were grown to approximately 85% confluence, then serum starved for 4 hours prior to treatment with stimuli or compounds. Sample Preparation Cells were lysed and samples collected according to the MILLIPLEX map Cell Signaling Buffer and Detection Kit (EMD Millipore Cat. No. 48-602) instructions. Microspheres We developed the MILLIPLEX map Akt/mTOR 11-plex Panel by conjugating specific capture antibodies to microsphere beads purchased from Luminex Corporation. Each set of beads is distinguished by different ratios of two internal dyes, yielding a unique fluorescent signature to each bead set. Human and Mouse tissue Homogenization Frozen tissue samples were weighed and placed on ice. Samples were homogenized with 1 mL lysis buffer per 50-100 mg mouse tissue or 1 mL lysis buffer per 30-50 mg of human tissue. Tissues were homogenized using the Omni International General Laboratory Homogenizer with Omni Tip- Plastic Generator Probes at setting level 2 for 30 seconds. Samples were then incubated with gentle rocking at 4 C for 15 minutes and centrifuged (10,000 g for 10 minutes at 4 C) to separate connective tissue, fat, ECM, etc. Supernatants were placed in new tubes. Protein concentration in each sample was determined
A. IP/Westerns
pp70S6K pIRS1 pGSK3a pIGF1R pGSK3b

by bicinchoninic acid (BCA) assay of sample aliquots we observed typical protein recoveries of 5-10% of initial tissue mass. After diluting samples to 2 mg/mL in lysis buffer, they were transferred to 96-well plates in preparation for assay with the MILLIPLEX map kit. Immunoassay Protocol The multiplex assay was performed in a 96-well plate according to product instructions supplied for the MILLIPLEX map Akt/mTOR 11-plex Panel (EMD Millipore Cat. No. 48-611). The plate was first rinsed with 100 L assay buffer. 25 L of controls and samples and 25 L beads were added to each well. Plates were incubated overnight at 4 C (alternatively can be incubated 2 hours at room temperature (RT)). Beads were washed twice with assay buffer, then incubated 1 hour at RT with biotinylated detection antibody cocktail. The detection antibody cocktail was replaced with 25 L streptavidin-phycoerythrin (SAPE) and incubated for 15 minutes at RT. 25 L of amplification buffer was added and incubated another 15 minutes at RT. Finally, the SAPE/amplification buffer was removed and beads were resuspended in 150 L assay buffer. The assay plate was read and analyzed in a Luminex 200 system. This is a compact unit consisting of an analyzer, a computer, and software (Luminex Corporation, Austin, TX).

results and Discussion


The MILLIPLEX map Akt/mTOR 11-plex Assay provided high specificity, indicated by the detection of proteins at the expected molecular weights as shown by immunoprecipitation/Western blot (Figure 2A). Specificity was also demonstrated by detection of the correct isoforms of GSK3a/b and IR/IGF1R (Figure 2B). In addition, analytical validation experiments (data not shown; details available on www.millipore.com) demonstrated high signalto-noise ratios, sample linearity, and precision, lending support to the robustness of this kit. All analytes in the MILLIPLEX map Akt/mTOR 11-plex Panel were detected in human and mouse tissues using the kit, with the exception of phospho-IGF1R, which is human-specific. Of interest is the observation that both breast cancer patients exhibited greater than a 2-fold increase in phosphorylation of mTOR compared to breast tissue from healthy subjects (Figure 3). This observation is consistent with cancer cells exhibiting a higher level of protein synthesis than normal cells. However, Akt,

NT EGF (A431)

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pAkt

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Figure 1. Specificity of the MILLIPLEX map Akt/ mtOr 11-plex Panel. Phosphorylated proteins were simultaneously detected in different cell lines treated with either insulin or IGF1. (A) Immunoprecipitation (IP) of phosphoproteins were performed with capture beads and detected by Western blotting with the biotinylated detection antibodies. (B) Isoform cross-reactivity tests were performed using human recombinant GSK3a/b by Western blotting or IR/IGF1R by IP/Western blotting. 14

NT Ins (HEPG2)

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which is upstream of mTOR, and p70S6K and RPS6, both downstream of mTOR, did not exhibit a significant change in phosphorylation levels.

HEK293 NIH3T3/IGF1R NIH3T3/IR IGF1 PIGF1R (IP/Western)

The addition of wortmannin, an inhibitor of PI3K, resulted in decreased levels of phosphorylation of several

downstream targets such as Akt, mTOR, p70S6K and RPS6 (Figure 4). Rapamycin, an mTOR inhibitor, also inhibited its downstream targets p70S6K and RPS6. Consequently, these studies demonstrate that tumor development is complex and involves more than the dysregulation of a
A. Phosphorylated Akt/mTOR proteins in human breast tissue samples
600 500
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single pathway. This conclusion further underscores the importance of simultaneous measurement of multiple phosphoprotein targets and demonstrates the utility of the MILLIPLEX map Akt/mTOR 11-plex Panel.

B. Phosphorylated Akt/mTOR proteins in mouse tissue samples


Mouse Tissues p70S6K Colon Pancreas Pituitary Prostate Skeletal Muscle Testicles Liver ND 305 28 143 186 250 80 IRS1 GSK3a 27 116 171 326 665 108 130 23 198 112 35 38 101 565 IGF1R GSK3b ND ND ND ND ND ND ND 42 62 176 176 552 57 51 Akt ND 215 198 163 15 52 226 PTEN 11094 10050 10956 10906 6643 13303 13630 IR 53 136 60 82 12 122 101 RPS6 7 676 967 345 265 133 5696 TSC2 4 212 540 454 374 1346 562 mTOR ND 81 1183 1102 636 1513 458

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Mouse Tissues Colon Pancreas Pituitary Prostate Skeletal Muscle Testicles p70S6K 157 188 2173 1556 1324 1222 1146 IRS1 GSK3a 61 87 5434 5382 4181 4241 5387 25 35 2722 2673 2704 2232 2421 IGF1R ND ND ND ND ND ND ND GSK3b 59 37 269 233 749 619 160 Akt 84 140 2212 2309 3475 3091 2145 PTEN 51 51 1752 1771 2035 1821 794 IR 163 220 509 475 528 426 525 RPS6 39 50 241 214 92 85 54 TSC2 86 82 370 367 789 608 240 mTOR 50 61 305 317 228 171 246

Phosphorylated Protein

Breast normal 1 Breast cancer 1 Breast normal 2 Breast cancer 2

15000 MFI 10000 5000 0 PTEN RPS6

Liver

Figure 3. Phosphorylated Akt/mTOR pathway proteins were simultaneously detected in human (A) and mouse (B) tissue samples. (20 g/mL). Total Akt/mTOR proteins were detected simultaneously in mouse (C) tissue samples using the MILLIPLEX map Total Akt/mTOR 11-plex Panel (available late 2011). Human matched breast normal and cancer tissue samples were purchased from Asterand. Mouse tissues from C57BL/6J males were purchased from Jackson Laboratory. Values were backgroundsubtracted and reported as mean fluorescence intensity (MFI). ND represents not detectable. Figure 4. Phosphorylated Akt/mtOr pathway proteins were detected simultaneously in HepG2 cells treated with various inhibitors. Cells were pretreated with 0.1 M wortmannin, 0.1 M rapamycin, 10 M U0126 (MEK1/2 inhibitor), 50 M LY-294002 (PI3K inhibitor), or Ro-31-8220 (PKC and GSK3b inhibitor) for 30 minutes prior to the addition of 10 g/mL insulin for 15 minutes. Values were background-subtracted and reported as percent of vehicle and insulin-treated values, respectively. references 1. Khan, I et al. Assay and Drug Dev Tech 2010, 8(1), 27-36. 2. Alvarex, R et al. G. J Clin Oncology 2010, 28(20), 3366-79. 3. Crowell, J et al. Mol Cancer Ther. 2007, 6(8), 2139-48. 4. Wang, X and Proud, C. Physiology 2006, 21, 362-9. 5. Vignot, S et al. Annals of Oncology 2005, 16, 525-37.

150 % Control Signal 125 100 75 50 25 0 IR (Tyr1162/ Tyr1163) RPS6 (Ser235/ Ser236) IGF1R (Tyr1135/ Tyr1136) mTOR (Ser2448) p70S6K (Thr424) Akt (Ser473) PTEN (SER380) IRS1 (Ser312) TSC2 (Ser939) GSK3 (Ser21) GSK3 (Ser9)

Wortmannin Rapamycin U0126 LY-294002 Ro-31-8220

Phosphorylated Protein

RELATED PRODUCTS
Available from www.millipore.com.
Description Catalogue No. Description Catalogue No.

MILLIPLEX map 11-plex Akt/mTOR Panel MILLIPLEX map Cell Signaling Buffer and Detection Kit IR (Tyr1162/Tyr1163) MAPmate IRS1 (pan Tyr) MAPmate Akt (Ser473) MAPmate PTEN (Ser380) MAPmate TSC2 (Ser939) MAPmate mTOR (Ser2448) MAPmate GSK3b (Ser9) MAPmate

48-611 48-602 46-688 46-627 46-677 46-679 46-691 46-686 46-690

p70S6K (Thr424) MAPmate Total IR MAPmate Total Akt MAPmate Total PTEN MAPmate Total mTOR MAPmate Total GSK3b MAPmate Total p70S6K MAPmate FlowCellect PI3K-mTOR Signaling Cascade Kit FlowCellect PI3K Activation Dual Detection Kit

46-629 46-687 46-675 46-678 46-685 46-689 46-630 FCCS05210 FCCS025105

15

Immuno-monitoring Using the Scepter 2.0 Cell Counter and Software Module
Amedeo Cappione, Ph.D.1; Emily Crossley2; Nagaraja thirumalapura, DVM, Ph.D.3; and Debra Hoover, Ph.D.1
1

EMD Millipore; 2Dept. of Pathology, Dept. Microbiology & Immunology, university of texas Medical Branch; Dept. of Pathology, university of texas Medical Branch

Abstract
Biological samples such as primary isolates or cultured cells are often heterogeneous mixtures of cells that differ by type and/or function. Such differences are most commonly determined by multicolor fluorescent antibody detection of specific cell markers using flow cytometry. In addition to variations in protein expression, many cell types and physiological states are also distinguishable by size. The ability to identify subsets on the basis of phenotypic differences and determine their relative frequencies (and concentrations) is critical to many aspects of research.

Materials and Methods


Human blood sample prep Human PBMCs were isolated from whole blood of healthy donors by Ficoll-Paque density centrifugation (GE Healthcare). Briefly, 9 mL of blood was diluted to 25 mL with phosphate-buffered saline (1X EmbryoMax PBS, EMD Millipore) and layered over 15 mL of Ficoll. Samples were centrifuged at 400 x g for 30 minutes with no brake, and the resulting PBMC layer was recovered. The PBMC fractions were washed twice with PBS prior to analysis. Scepter cell counting

The Scepter cell counter combines the ease of automated instrumentation and the accuracy of impedance-based particle detection in an affordable, handheld format. Using a sensor with a 40 m aperture, the Scepter cell counter can accurately and precisely count a broad range of cell types, including small cells (> 4 m in diameter) and peripheral blood mononuclear cells (PBMC)1. This article outlines three examples of experiments using the Scepter cell counters sensitive size-discriminating capability to demonstrate rapid, qualitative assessment of individual cell population frequencies in complex cell mixtures.

The Scepter cell counter was used to count samples following the simple on-screen instructions. Briefly, the user attaches a 40 m sensor, depresses the plunger, submerges the sensor into the sample, then releases the plunger drawing up 50 L of cell suspension. The Scepter cell counter detects each cell passing through the sensors aperture, calculates cell concentration, and displays a histogram of cell diameter or volume. Test files were uploaded and analyzed using Scepter Software Pro to determine the concentrations and relative cell frequencies for the lymphocyte and monocyte fractions. Cell counting and viability determination using Guava ViaCount assay 10 L samples were mixed with 190 L ViaCount reagent, incubated for 5 minutes at room temperature (RT). Sample data was acquired on a guava easyCyte instrument and analyzed using guava ExpressPro software. Cell surface staining and subset determination For each sample,100,000 PBMCs were resuspended in 100 L PBS+0.1% bovine serum albumin (BSA). Samples were stained with the following combination of fluorescently labeled antibodies: CD3-PE (T cells), CD19-Alexa Fluor 488 (B cells), CD16/CD56-APC (NK cells), and CD14- PECy7

01

ExamplE 1: Lymphocyte vs. monocyte subset discrimination in human PBMCs

Introduction
The human immune system is comprised of cell subsets with distinct functional profiles that fight pathogens. Assessing profiles of the various subsets, such as lymphocytes and monocytes, can help identify molecular signatures that may facilitate research, ranging from vaccine development to prognostic advancements.The Scepter cell counter, in combination with Scepter Software Pro, provides a tool for rapid determination of lymphocyte and monocyte concentrations in PBMC isolates.

16

(Monocytes) (antibodies all from eBioscience). Single stain and isotype controls were included to ensure proper instrument setup. Samples were incubated at RT for 20 minutes, washed twice with PBS, then resuspended in 200 L PBS prior to acquisition. Samples were analyzed (3000 cells/sample well) on a guava easyCyte system using guava ExpressPro software.

particle size. While overlap exists between dead cells and debris, live cells constituted a fraction made up of two differently-sized cell types. PBMC subset enumeration (t cells, B cells, NK cells, monocytes) PBMC can be subdivided into many distinct cell types based on expression of specific surface markers. The abundance and relative distribution of these subsets are functions of developmental state as well as overall health. Two main PBMC populations are lymphocytes (CD14-) and monocytes (CD14+). Lymphocytes can be subdivided into T cells (CD3+), B cells (CD19+), and NK cells (CD16/56+). To examine the ability of 40 m sensors to discriminate lymphocytes from monocytes, PBMC fractions were analyzed by both a guava easyCyte and Scepter cell counter. Representative plots are presented in Figure 3. In each case, three main peaks were identified by each analysis method, with greater peak resolution in the flow cytometry data, particularly in the separation of lymphocytes from debris. The peaks correspond to lymphocytes (small cells), monocytes (large cells), and a debris/dead cell fraction.

results
Viability assessment of PBMC Freshly prepared or frozen PBMC samples consist of live cells, dead cells, and a considerable amount of cellular debris. To understand the relative proportions, freshly isolated PBMC were stained with ViaCount reagent. The reagent consists of a cell-permeant nuclear dye that stains all nucleated cells, and a cell-impermeant dye that brightly labels dying cells. Cellular debris is not stained by either dye. In the example shown, more than 95% of the cells were viable. The three components were also distinguishable by particle size. The histograms in Figure 1B show the distribution of particles in the different fractions as function of forward scatter, a flow-based correlate of

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Figure 1. Size-based discrimination of live vs dead cells. (A) live cell (blue), dead cell (red), and debris (green) fractions were defined using ViaCount reagent. (B) The four histograms show the relative distribution of total events and each gated fraction.

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CD19+

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CD16/56+

Figure 2. Multicolor flow cytometric analysis of PBMC fractions. (A) Live cells (red) are distingui shed from debris/ dead cells (green). (B) Live cells are fractionated based on expression of CD14, CD3, CD16/56, and CD19. (C) The four histograms show the localization of each subset to either of the two peaks defined by forward scatter.

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17

Flow Cytometry

Scepter (40 m) test Cell Fraction Lymphocyte Monocyte 2 Lymphocyte Monocyte Lymphocyte Monocyte 4 Lymphocyte Monocyte 5 6 Lymphocyte Monocyte Lymphocyte Monocyte 7 8 9 Lymphocyte Monocyte Lymphocyte Monocyte Lymphocyte Monocyte

relative Frequency Scepter1 58 42 68 32 66 34 62 38 64 36 62 38 65 35 59 41 64 36 Forward Scatter2 65 35 72 28 69 31 67 33 66 34 58 42 72 28 61 39 72 28 Staining3 63 37 71 29 71 29 64 36 67 33 60 40 72 28 61 39 72 28

Count

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Figure 3. Three examples comparing histogram plots for human PBMC samples acquired on the Scepter cell counter (diameter) and guava easyCyte (forward scatter) platforms. Plots derived from both platforms demonstrate three distinct peaks corresponding to lymphocyte, monocyte, and dead cell/debris fractions. The difference in counts displayed (Y-axis) is due to differences in sample dilution between the guava flow cytometer and the Scepter cell counter.

table 1. Subset frequencies from nine PBMC samples. Samples were analyzed using the guava easyCyte and Scepter platforms. 1 Values were derived from diameter plots. 2Values were derived from FSC histograms using guava easyCyte. 3Frequencies were derived as follows: Lymphocyte = % T cells + % NK + % B cells; Monocytes = % CD14+ cells

Across the nine PBMC samples analyzed (Table 1), the average mean cell diameters were 7.230.30 m and 10.020.20 m for lymphocytes and monocytes, respectively. Values were consistent with previously reported size ranges2. Cell frequencies were also determined by three methods: Scepter diameter plot, guava easyCyte forward scatter, and antibody staining. The Scepter values slightly underestimated the lymphocyte fraction while overestimating the monocyte subset. This is likely the result of the Scepter cell counters comparatively lower resolution as well as subjectivity and user bias in gating. Overall, there was good agreement between the different analytical techniques with values varying by <10% in nearly all cases.

culminating in the production of specifically-tuned immune responses. Elevated CD25 levels are a hallmark of late-stage T cell activation3. Assays measuring changes in immune cell activation are commonly used to identify patterns of immune response in clinical diagnostics and therapeutic development. The in vivo T cell response can be mimicked ex vivo by binding T cells to immobilized anti-CD28 and anti-CD3 antibodies4. PBMCs can also be activated through exposure to more generic inducers of cell proliferation, such as the plant lectin phytohemagglutinin (PHA). Ex vivo activation enables detailed studies of the molecular mechanisms regulating T cell activation and response.

02

ExamplE 2: Human T cell activation

Materials and Methods


Cell culture/activation PBMC fractions were isolated as previously described. Initial cell concentrations were determined using the Scepter cell counter. All cultures were performed in RPMI 1640 supplemented with 10% fetal bovine serum (FBS). PBMC (400,000 cells/mL, 24-well plates) were stimulated for two days in the presence of soluble anti-CD28 mAb (clone 28.2, BD Pharmingen, 2 g/mL) on plates pre-coated

Introduction
In vivo, T cells are activated through binding of the T cell receptor to antigen-presenting cells. In response to stimulation, T cells undergo numerous phenotypic changes, including increased cell size, secretion of cytokines, and up-regulation of CD25 surface expression, ultimately
18

with anti-CD3 mAb (clone HIT3a; BD Pharmingen, 10 g/ mL). Mitogenic stimulation was carried out in the presence of 2 g/mL PHA (Sigma). Unstimulated control cultures were also analyzed. Scepter cell counting Sample acquisition and data analysis was performed as previously described. Test files were analyzed using Scepter Software Pro to determine the degree of cell activation as well as concentrations for both unstimulated and activated fractions. CD25 staining for activation Following two-day stimulation, cultures were harvested, washed twice with PBS, counted, and stained as previously described. For each culture, 100,000 cells were resuspended in 100 L PBS+0.1% BSA. To distinguish the activated T cell fraction, samples were stained with anti-CD3-PE ( T cells) and anti-CD25-APCeFluor780 (eBioscience). Samples were analyzed (3000 cells/sample well) on a guava easyCyte HT system using guava ExpressPro software.

results
To evaluate utility of the Scepter cell counter for rapid qualitative monitoring of immune cell activation, PBMCs were stimulated in culture using two different mechanisms: (1) CD3/CD28 Ab-mediated co-stimulation of the TCR and (2) stimulation by PHA. CD25 expression correlated with an increase in cell size (Figure 4A). Figure 4B shows three main populations of cells: resting T cell (CD3+/CD25-), activated T cells (CD3+/CD25+), and non T cells (CD3-/CD25-). By comparison, cultures stimulated with CD3/CD28 Ab showed greater levels of T cell activation than those exposed to PHA. Control cultures displayed very low frequencies of activated cells.. Figure 4C-D compares the Scepter cell counters ability to detect activation to that of the guava easyCyte platform. While the Scepter cell counter clearly detected the larger, activated cell fraction in stimulated cultures, the device was unable to simultaneously discriminate the unstimulated population. This was most likely due to the presence of a large number of overlapping dead cells

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Figure 4. CD25 expression correlates with size increase. (A) An elliptical gate was used to identify live cells. CD25+ cells are red. Live cells are fractionated using CD3 and CD25 Ab. (B) The two histograms in each row show the relative distribution of each fraction as a function of particle size (forward scatter). Plots are based on total events and live cells, respectively. (C) Scepter histogram data for each sample. 19

(green cells, Fig. 4A). This was clearly demonstrated in the untreated sample--two peaks were seen in the histogram plot of total cells (Marker R5). By comparison, the live cellonly histogram showed a single peak within R5.

Mice were sacrificed on day 30 post-infection and spleens harvested. Single-cell suspensions were prepared using the GentleMACS Tissue Dissociator following the manufacturers instructions (Miltenyi Biotec Inc., CA). Determination of splenocyte cell distribution using the Scepter cell counter Cells were diluted (~1 2 x 105 cells/mL) and acquired using the Scepter cell counter and 40 M sensor. Gates were set to exclude red blood cells using an uninfected, nave mouse sample. Splenocytes from three uninfected mice and three mice infected with Ehrlichia muris were used. Data were analyzed using Scepter Software Pro. Determination of splenocyte cell distribution by flow cytometry Splenocytes were incubated with Near IR LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen, CA) for 10 minutes in PBS at RT. Cells were washed, blocked with Fc Block (BD Biosciences, CA), purified rabbit, rat, and mouse IgGs (Jackson ImmunoResearch, PA) then stained with antiB220-PerCPCy5.5 (BD Biosciences, CA) and anti-Ter119APC (eBioscience, CA) and analyzed by flow cytometry.

03

ExamplE 3: Splenic cell shift in murine model of Ehrlichiosis

Introduction
Human monocytotropic ehrlichiosis (HME) is a tick-borne disease caused by the obligately intracellular pathogen Ehrlichia chaffeensi. HME manifests as nonspecific flulike symptoms but can progress to life-threatening toxic shock-like syndrome with anemia, thrombocytopenia, and multi-organ failure. Extensive inflammation in the absence of bacterial burden suggests that mortality is due to unregulated Immunopathology5. In this study, a murine HME model based on Ehrlichia muris infection of C57BL/6 mice was used to investigate the immune response, in particular, changes to splenocyte population dynamics.

Materials and methods


Infection of mice with Ehrlichia muris Six- to eight-week-old C57BL/6 mice were infected intraperitoneally with Ehrlichia muris (~1 x 104 genomes).

B
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C
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Large Cells Lymphocytes

Untreated

B200-PerCP-Cy5.5

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SSC

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Small Cells Erythrocytes

Infected

Count

Diameter (m)

FSC

Ter119-APC

FSC

Figure 5. Forward vs. side scatter plots reveal the presence of two main splenocyte populations. (A,B) The fraction containing larger cells is comprised of leukocytes (B, NK, and T cells), small cells are predominantly erythrocytes (Ter119+). (C,D) Three distinct peaks can be visualized by flow cytometry. For the Scepter cell counter, the size of the debris peak is below the minimum particle diameter for detection.

20

results
Mouse splenocytes were analyzed by multicolor flow cytometry (Fig. 5). Size-based discrimination confirmed the presence of two distinct cell populations in all samples. Staining with antibodies specific for B cells (B220) and erythrocytes (Ter-119) demonstrated that erythrocytes were restricted to the subset containing smaller cells, while the fraction of large cells was composed primarily of lymphocytes. Flow-based histograms showed three distinct populations corresponding to debris, lymphocytes (large cells) and erythrocytes (small cells). Analysis of splenic cell isolates from infected and control mice revealed an overall shift in the relative cell distribution. Specifically, infected samples showed an increase in % erythrocytes when compared to the lymphocyte subset (Fig. 5C and Table 2). In contrast, the Scepter cell counter was able to detect only two peaks; the debris fraction was smaller than the minimum particle diameter for detection. The erythrocytes fraction was also at the limits of detection potentially skewing the subsets count. However, the Scepter cell counters quantitation of splenocyte counts and percentages were in very good agreement with the values determined by flow cytometry. An assessment of cell concentration values indicated the shift was due to an expansion of splenic erythrocytes while lymphocyte values remained constant (Table 2). These findings were consistent with previously published results5.

and measuring differences in the relative frequencies (and concentrations) of multiple cell types in samples is very useful for accelerating research in these fields. .We have presented data indicating that the guava easyCyte benchtop flow cytometer and the Scepter cell counter can determine such differences in fresh primary isolates and cultured samples. While the flow cytometer is the more sophisticated and quantitative tool, the Scepter cell counter offers rapid, on-line sample qualification and serves as an adjunct to existing methods. The ability of the Scepter device to ensure reproducible cell counts improves data quality during experimental setup and downstream cell-based analyses.

references 1. Smith, J et al. The New Scepter 2.0 Cell Counter Enables the Analysis of a Wider Range of Cell Sizes and Types With High Precision. EMD Millipore Cellutions 2011 Vol. 1: p 19-22. 2. Daniels, V. G., Wheater, P. R., & Burkitt, H. G. (1979). Functional Histology: A Text and Colour Atlas. Edinburgh: Churchill Livingstone. ISBN 0-443-01657-7. Prager, E. et al. (2001) Induction of Hyporesponsiveness and Impaired T Lymphocyte Activation by the CD31 Receptor: Ligand Pathway in T-Cells. J. Immunol. 166: 2364-2371. Levine, B. L., et al. (1997). Effects of CD28 Costimulation on Long-term Proliferation of CD4+ T-cells in the Absence of Exogenous Feeder Cells. J. Immunol. 159:5921 5930. MacNamara, K.C., et. al. Diminished Hematopoietic Activity Associated with Alterations in Innate and Adaptive Immunity in a Mouse Model of Human Monocytic Ehrlichiosis. Infect. And Immunity. 2009; 77:4061-69.

3.

4.

5.

Overall Conclusions
Evaluation of immune responsiveness to viral, bacterial, and environmental exposure is an important tool in for determining pathogenicity and toxicity. The availability of simplified methods for identifying activation states

Flow test Sample Control-1 Control-2 Control-3 Infected - 1 Infected - 2 Infected - 3 Erythrocyte 16 21 14 42 29 38 Lymphocyte 84 79 86 58 71 62 Erythrocyte 27 29 22 40 30 33

Scepter Lymphocyte 73 71 78 60 70 68

Concentration (x105) Erythrocyte 1.86 2.61 2.17 4.3 2.82 3.5 Lymphocyte 4.65 5.54 5.78 5.22 4.93 5.84

table 2. Subset frequencies from six splenocyte isolates. Aliquots from each sample were analyzed by flow cytometry and the Scepter cell counter. Cell concentrations were derived on the using the Scepter cell counter.

21

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Available from www.millipore.com.

Scepter 2.0 Cell Counter


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Guava easyCyte Benchtop Flow Cytometers


Description High-throughput Sampling Instruments guava easyCyte 8HT Base System guava easyCyte 6HT/2L Base System guava easyCyte 5HT Base System PCA-96 Base System Single Sampling Instruments easyCyte 5 Base System easyCyte 6-2L Base System easyCyte 8 Base System 1 1 1 0500-5005 0500-5007 0500-5008 1 1 1 1 0500-4008 0500-4007 0500-4005 0100-8710 Quantity Catalog No.

22

Mitochondrial to Nuclear DNA Ratio: Sensitive Biomarker of Stem Cell Differentiation


Jason C. Poole, tony Gaige, Jackie Stupack, Nicole A. Leonetti, and Adrin Vilalta EMD Millipore

Introduction
Mitochondria numbers change in response to numerous stimuli and cellular conditions including drug toxicity, caloric intake, cancer (Warburg effect) and differentiation, among others1,2,3. Here, we describe an assay that can determine mitochondrial levels accurately and easily using quantitative PCR (qPCR). The assay generates fluorescent signals for two mitochondrial- and two nuclear-encoded genes; changes in the ratio of mitochondrial to nuclear signals correlate to changes in mitochondrial levels. Importantly, the qPCR primers were designed and validated not to amplify nuclear sequences (mitochondrial pseudogenes). In vitro differentiation of stem cells requires the development of finely-tuned differentiation protocols. Monitoring changes in molecular biomarkers can provide a powerful tool to guide the development and execution of such protocols. Here we provide data to support the use of the mitochondrial to nuclear DNA ratio assay to monitor the toxic effects of ethidium bromide on osteosarcoma cells and to track the differentiation of stem cells to hepatocytes.

qPCr PCR primer pairs were pre-aliquoted in optically clear 96-well plates. DNA was amplified using a SYBR Greencontaining master mix and a fast block protocol. PCR method can be used with input DNA ranging from 0.2 to 20 ng. Calculation of relative Copy Numbers Ct values obtained for each of the four genes were used once to determine mitochondrial DNA copy numbers. This is accomplished by averaging the copy numbers calculated from the ND1/BECN1 and the ND6/NEB pairs. The Ct values from the ND1 gene are subtracted from the BECN1 Ct to obtain Ct1. Likewise; ND6 Ct is subtracted from NEB to obtain Ct2. To calculate copy number, we used the average of Set 1 = (ND1/BECN1) and Set 2 = (ND6/NEB) ratios. To determine the individual ratios from set 1 and set 2 the following calculation was used: N = 2Ct where Ct1 = CtNuc1-CtMito1 and Ct2 = CtNuc2-CtMito2.

rho Zero Cell Line 143B rho zero cells (143B 0; cells devoid of mitochondria) were created by growing 143B cells (human osteosarcoma cells, TK-) in their standard media (MEM, 5% FBS, 100 g/ml 5-bromo-2-deoxyuridine) in the presence of 50 ng/mL ethidium bromide to achieve 0 status and 50 g/mL uridine, which is needed for cell viability once cells reach glycolytic state. Approximately two months after start of ethidium bromide treatment (8 passages), cells achieved full 0 status and remained stable post removal of ethidium bromide from the media. Stem Cell Lines A highly characterized proprietary human ESC line and differentiated hepatocytes were kind gifts from California Stem Cell (Irvine, CA).

Methods
Primer Design and Validation PCR primers specific to two human mitochondrial (ND1 and ND6) and two nuclear genes (BECN1 and NEB) were designed to have closely matched amplification efficiencies. Extensive bioinformatics analysis was performed to identify regions in the mitochondrial genome without any significant homology in the nuclear genome. These regions were used to design PCR primers for the mitochondrial genes. Total DNA from either human osteosarcoma 143B cells or a derivative lacking mitochondria (143B 0) was used to calibrate the assay. Both 143B and 143B 0 generate the same Ct values for nuclear genes; mitochondrial genes can be detected in the 143B but not in the 143B cell lines.
0

23

BECN1BECN1 Standard Curve Standard Curve


33.0 33.0 32.5 32.5 32.0 32.0 31.5 31.5 31.0 31.0 30.5 30.5 30.0 30.0 29.5 29.5 29.0 29.0 28.5 28.5 28.0 28.0 27.5 27.5 27.0 27.0 26.5 26.5 26.0 26.0 25.5 25.5 25.0 25.0 24.5 24.5 24.0 24.0 23.5 23.5 23.0 23.0 0.01 0.020.01 0.02 0.2 0.1 0.2 1 0.1

2 31 2 10 45 30 20 30 45 3 20 10 100

31 31 30 30 29 29 28 28 27 27 26 26 25 25 24 24 23 23 22 22 21 21 20 20 19 19 18 18 17 17 16 16 15 15 14 14 13 13 100 0.0001 0.0001 0.002 0.01 0.020.01 0.02 0.11 2 345 1 10 2030 10 2030 100 0.001 0.001 0.002 0.1 0.2 0.2 2 345 100

ND1 Standard Curve Curve ND1 Standard

results
qPCr Assay Dynamic range for mtDNA and Nuclear Genes Dynamic range of the assay was determined by testing input total DNA from 0.002 to 20 ng (Figure 1). A linear response was observed for the whole input range in the case of mitochondrial genes; nuclear genes generate a linear response from 0.2 to 20 ng of input DNA. Given these results we routinely used anywhere from 0.2 to 20 ng of total DNA to obtain accurate mitochondrial to nuclear DNA ratios. Mitochondria Number Estimate Normal cells are expected to have anywhere from 100 to about 10,000 copies of mitochondrial DNA, depending on cell type and cellular status. In a representative experiment, 2.5 ng of total DNA from 143B cells was tested in this assay producing Ct values of 16.12 for the mitochondrial gene ND1 and 25.12 for the nuclear gene BECN1. A value of 512 is found when calculating the ND1/BECN1 ratio as described in the methods section. A similar value can be obtained when calculating the ND6/NEB ratio. This value of 512 was consistent with expectations for a metabolically active cell. In contrast, when total DNA from the 143B 0 was tested, BECN1 produced a Ct value of 25 but no ND1 could be detected. This result was consistent with a cell line devoid of mitochondria (0). These results are summarized in Figure 2.

CT

CT

CT

Quantity Quantity NEB Standard Curve Curve NEB Standard


29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 2 31 2 10 45 30 20 30 45 3 20 10 100 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13

CT

Quantity Quantity ND6 Standard Curve Curve ND6 Standard

35 34 33 32 31 30

35 34 33 32 31 30

CT

CT

CT Quantity Quantity

28 27 26 25 24 23 22

28 27 26 25 24 23 22

0.01 0.020.01 0.02 0.2 0.1 0.2 1 0.1

12 12 100 0.0001 0.0001 0.002 0.01 0.020.01 0.02 0.11 2 345 1 10 2030 10 2030 100 0.001 0.001 0.002 0.1 0.2 0.2 2 345 100

CT

29

29

Quantity Quantity

Figure 1. qPCr assay dynamic range for nuclear and mitochondrial genes. Standard curves for nuclear (BECN1 and NEB) and mitochondrial (ND1 and ND6) plot the Ct values obtained with respect to varying amounts of input total DNA.

10 1 0.1

10 Flow Cytometry Analysis 1 The 143B 0 cells were tested using the FlowCellect

MitoPotential Red Kit (EMD Millipore) for the guava


Rn

Rn

easyCyte benchtop flow cytometry system (Figure 3) in order to obtain confirmatory data on the mitochondrial
0.01

0.1

0.01 0.001 0.0001

0.001 status 0.0001

of the cell line. As expected, the analysis confirmed


4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

the extreme depolarization of the mitochondrial membrane potential in the 143B 0 line.Cycle
2

4 6

8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Cycle

10 1 0.1

Rn

0.01 0.001 0.0001

12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Cycle

4 6

8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Cycle

Figure 2. Mitochondrial DNA cannot be detected in 143B 0 line. Human mitochondrial to nuclear DNA assay was used to confirm the mitochondrial status of the 143B 0 line (B) as compared to the parental 143B cell line (A). Blue traces indicate the signal corresponding to the nuclear genes while the red and orange traces indicate mitochondrial genes signal.

24

A
104

B
104
10 1

103

103

0.1 0.01 0.001 0.0001 0.00001

Mitosense Red

Mitosense Red

102

102

101

101

100 0 10

101

102

103

104

100 0 10

101

102

103

104

Rn

0.000001

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 Cycle

7-AAD

7-AAD

Figure 3. Flow cytometry analysis confirms 0 status of negative control cell line. Mitochondrial content status for the parental 143B (A) and the 143B 0 cell lines was confirmed using flow cytometry, which showed that, while the parental cell line stained largely positive (top left quadrant, A) for intact mitochondrial membrane potential, the negative control cell line stained negative for mitochondrial membrane potential (bottom left quadrant, B).

Figure 4. Mitochondrial content changes in response to presence of ethidium bromide. 143B osteosarcoma cells were incubated in the presence of 50 ng/mL of ethidium bromide. Total DNA was obtained at different passages (0 to p7). Red trace indicates fluorescent signal corresponding to the nuclear genes; green trace represents mitochondrial genes signal (2 ng total DNA input).

Monitoring Mitochondrial toxicity Ethidium bromide preferentially accumulates in the mitochondria thus damaging mtDNA at a faster rate compared to nuclear DNA. Consequently, culture in the presence of low levels of ethidium bromide (~50 ng/mL) is used routinely to generate cell lines. Figure 4 showed a
0

1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0

decrease in mitochondrial content in A549 (lung epithelial carcinoma) cells with respect to passage number in the presence of ethidium bromide. Stem cell differentiation results in significant change in mitochondrial content Total DNA from well-characterized human embryonic stem cells (hESC) was analyzed for relative levels of mtDNA and compared to fully differentiated hepatocytes obtained from the hESCs. A statistically significant increase in relative mtDNA content was observed upon differentiation (Figure 5).
RQ

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

Conclusion
Mitochondrial content is closely linked to the metabolic state of the cell; thus, changes in the number of mitochondria are closely connected to cellular responses to stress, energy homeostasis and disease1. Moreover, mitochondria numbers have been shown to change upon differentiation2,3.

Hepatocyte

Figure 5. Differentiation of human embryonic stem cells (hESC) results in increase in mitochondrial content. Relative levels (RQ) of mitochondria were determined both in well-characterized embryonic stem cells and compared to those observed in hepatocytes. hESC

25

For these reasons, it is important to have reliable, sensitive, specific method to measure the mitochondrial to nuclear DNA ratio when tracking these cellular processes. Determination of mitochondria levels in the cell can be estimated using biochemical and immunological approaches; however, these methods usually require large samples and may lack the sensitivity to detect small changes in mitochondrial content. The assay discussed here provides an alternative approach based on sensitive qPCR methodologies that only require small amounts of sample. Data generated with the mitochondrial to nuclear DNA ratio qPCR assay are consistent with data obtained using other approaches such as multiplex immunoassays. The qPCR assay can be used to monitor changes driven by mitochondrial toxicity. Clearly, this application should prove useful in drug toxicity screens. Data presented here also demonstrate that the assay is useful in monitoring mitochondrial changes driven by stem cell differentiation.

Given the central role of mitochondria in cellular health the mitochondrial to nuclear DNA ratio qPCR assay should prove useful to scientists in a wide variety of fields such as cancer, metabolic diseases, drug development and stem cell research.
references 1. Clay Montier LL et al. Number matters: control of mammalian mitochondrial DNA copy number. J Genet Genomics. 2009 Mar;36(3):125-31. 2. St John JC et al. Mitochondrial DNA transmission, replication and inheritance: a journey from the gamete through the embryo and into offspring and embryonic stem cells. Hum Reprod Update. 2010 Sep-Oct;16(5):488-509. 3. Facucho-Oliveira JM et al. Mitochondrial DNA replication during differentiation of murine embryonic stem cells. J Cell Sci. 2007 Nov 15;120(Pt 22):4025-34.

RELATED PRODUCTS
Available from www.emdbiosciences.com.
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NovaQUANT Human Mitochondrial to Nuclear DNA Ratio Kit Available from www.millipore.com.
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72620

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FlowCellect MitoPotential Red Kit guava easyCyte 8HT Base System

FCCH100105 0500-4008

26

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ESGrO-2i Medium
Maintain ground state pluripotency and promote growth at clonal density of iPS and mouse ES cells with ESGRO-2i, a defined medium formulated with EMD Millipores gold standard ESGRO medium supplement, a highly potent formulation of mouse leukemia inhibitory factor (LIF), and provided with GSK3b and Mek1/2 inhibitor supplements. In addition to promoting mouse ES cell culture and derivation, 2i/LIF-based media have been shown to promote partially reprogrammed cells to a fully reprogramming nave pluripotent state1.
reference: 1. Silva, J et al. Promotion of Reprogramming to Ground State Pluripotency by Signal Inhibition. PLoS Biol. 2008; 6(10): e253. Description Qty Catalogue No.

ESGRO-2i maintains pluripotency of 129SvEv ES cell colonies. After 10 passages in ESGRO-2i medium, colonies express Oct4 (left, red) and SSEA-1 (right, red) with blue DAPI nuclear staining.

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Now Available: EpiGrO Human Corneal Epithelial Cells and Ocular Epithelia Cell Culture Complete Medium Kit Advance your studies of ocular irritation, toxicity, and drug development. Use EMD Millipores EpiGRO epithelial cells along with the EpiGRO complete media kits to experience more convenient, reliable, and consistent culture of your epithelial cells. Serum-free or low-serum formulations Improved viability and morphology Increased cell culture longevity and proliferation No phenol red or other additives that produce masking effects Unique light-blocking and temperature-monitored packaging to promote media stability and better results

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This new, ready-to-use expansion medium is optimized for the culture of human fibroblasts in an environment free of nonhuman animal components. This medium supports the growth of these cells in 2% human serum at rates equal to or greater than
Human fibroblasts grown in FibroGRO xeno-free expansion medium at Day 8.

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Human NK Cell Characterization Kit


conjugated antibodies, Anti-CD3-APC, Anti-CD56-PE, and Anti-CD16 FITC. Use this threecolor kit to characterize NK cells within mixed cell populations. The antibodies provided have been carefully titrated to ensure the ability to accurately measure the expression of all markers simultaneously.
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Human StEMCCA reprogramming Kits


Efficient iPS Cell Generation with a Single Vector Unlike traditional iPS generation, which requires simultaneous co-infection of Oct4, Klf4, Sox2 and c-Myc (OKSM) factors by four separate expression vectors, the STEMCCA kits use a single polycistronic lentiviral vector to improve efficiency and reduce the number of viral integrations. These kits include ready-to-use lentiviral particles containing a single vector expressing human OKSM genes from a single polycistronic transcript. Two formats are available, constitutive and Cre/LoxP regulated for excision of the exogenous reprogramming transgenes.

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29

Human iPS Cell Boost Supplement


Increase the quality and number of your human iPS cells with EMD Millipores new small molecule Human iPS Cell Boost Supplement. Theres no need to optimize your small molecule concentrations, each supplement is provided ready to use for supplementing 300 mL of medium.

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Human Oligodendrocyte Differentiation Kit


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Accurately characterize different stages of oligodendrocyte development with these validated antibodies, as well as antibodies for the detection of neurons and astrocytes. Markers detected by provided antibodies: NG2, PLP, GalC, MOG, MAP2, and GFAP.
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Track Cell Health and Differentiation With NovaQUANT Mitochondrial to Nuclear DNA Ratio qPCR Assays
Step ahead of the expanding mitochondrial research field with NovaQuANt assays, an innovative, easy way to detect mitochondrial genes with high specificity and sensitivity. These optimized qPCR assays accelerate studies of cellular homeostasis, influenced by differentiation, stress, metabolism, toxicity and disease.

Achieve greater specificity and reliability: Matched amplification efficiencies of mitochondrial and nuclear primer pairs Validated targets eliminate pseudogene bias Protocols optimized for sensitivity and linearity Included wild type and mitochondrial negative total DNA for high-specificity controls read the article (page 23 of this issue) on using NovaQuANt assays to monitor stem cell differentiation.

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31

Cellutions:

The Newsletter for Cell Biology Researchers


To subscribe to our quarterly Cellutions newsletter and read about the latest research, new products, and innovative protocols for revolutionizing cell culture and analysis, visit www.millipore.com/cellutions.

BRANCHING OUT IN STEM CELL RESEARCH


Partner with EMD Millipore to expand the scope and power of your stem cell research. Our stem cell scientists work with our customers to stay on the cutting edge of technology and bring you the best of whats new. Visit our stem cell site at www.millipore.com/stemcells to stay up to date.

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EMD Millipore, NovaQuant and the M mark are trademarks of Merck KGaA, Darmstadt, Germany. ESGRO, MILLIPLEX, Millicell, EmbryoMax, ViaCount and guava are registered trademarks of Millipore Corporation. STEMCCA, Scepter, FibroGRO, QCM, FlowCellect, guava easyCyte and EpiGRO are trademarks of Millipore Corporation. StemPRO, Alexa Fluor, and SYBR are registered trademarks of Life Technologies, Inc. mTeSR is a registered trademark of WiCell Research Institute. ATCC is a registered trademark of the American Type Culture Collection. Matrigel and FcBlock are trademarks of BD Biosciences. Geltrex is a trademark of Life Technologies, Inc. Luminex, xMAP are registered trademarks and Luminex 200 is a trademark of Luminex Corporation. Omni Tip is a trademark of Omni International. GentleMACS is a trademark of Miltenyi Biotec Inc. Lit No. PR3322EN00 BS-GEN-11-04628 Printed in the USA 6/2011 2011 Millipore Corporation. All rights reserved.