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Cheryl D. Helgason and Cindy L. Miller (eds.), Basic Cell Culture Protocols, Methods in Molecular Biology, vol. 946,
DOI 10.1007/978-1-62703-128-8_14, Springer Science+Business Media, LLC 2013
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1. Introduction
Macrophages are critical players in all aspects of the immune
response to foreign pathogens and tumor cells. Resident tissue macrophages are poised to respond to infection or injury and initiate an
inflammatory response to danger or pathogen associated molecular
patterns. Within 24 h of insult, monocytes are recruited from the
circulation and move into a site of injury or infection where they
mature into classically activated macrophages, also called killer or
M1 macrophages (1). These M1 macrophages amplify the innate
immune response producing cytokines and chemokines. They can
also present antigen to initiate the acquired immune response.
When the inflammatory insult has been dealt with, macrophages
remain at the scene and are converted by the local cytokine milieu
to participate in the resolution phase of inflammation. These macrophages participate in debris scavenging, angiogenesis, tissue
remodeling, and wound healing (2). These macrophages are called
alternatively activated, healer, or M2 macrophages (3).
M1 and M2 macrophages represent extremes of macrophage
polarization. Increasingly, we recognize that macrophages are heterogeneous both in their phenotype and functional responses to
inflammatory stimuli (4). In complex systems, this may be due to
multiple, simultaneous stimuli acting on individual cells, intermediate or transitional phenotypes, as well as the effects of populations of macrophages. Despite this, it is still critical to define
features of polarized macrophages to enable comparison and categorization of macrophage phenotype and function to better understand their role in normal and pathophysiologies. M1 macrophages
are activated by IFNg or LPS and produce robust amounts of reactive oxygen species and pro-inflammatory cytokines (IL-12, TNFa,
IL-23) and chemokines and murine M1 macrophages upregulate
inducbile nitric oxide synthase (iNOS) to produce the reactive
nitrogen species, nitric oxide (NO) (1). The canonical M2 macrophages, also referred to as M2a macrophages, are activated by
IL-4 or IL-13 and their activation can be enhanced by co-treatment with IL-10. In response to inflammatory stimuli, these macrophages produce lower amounts of pro-inflammatory cytokines
and higher amounts of anti-inflammatory IL-10 relative to their
M1 counterparts. Additionally, murine M2 macrophages up-regulate expression of arginase I (argI), Ym1 (a mammalian chitinase),
and FIZZ1 (also known as RELMa) (5).
A critical switch that defines murine macrophage activation
and polarization is the way in which the cells metabolize L-arginine
(6, 7). Murine M1 macrophages metabolize L-arginine by iNOS
to produce NO. NO is a reactive nitrogen intermediate that can
damage DNA, thereby killing foreign microorganisms or tumor
cells and also causes host tissue damage. M2 macrophages
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2. Materials
2.1. Tissue Culture
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2.3. SDS-PAGE
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2.6. NO Assay
2.7. Enzyme-Linked
Immunosorbent
Assays
3. Methods
3.1. Tissue Culture
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palm firmly several times. Ensure that cells have lifted off of the
flask by examining the flask under the microscope. Remove
resuspended cells into a 15 ml conical Falcon tube and wash
the flask with an additional 10 ml of IMDM, 10% FBS. Pool
and spin down the cells at 300 g for 5 min. Resuspend cells in
a small volume and count viable cells using a hemocytometer.
9. Cells can be harvested into the appropriate buffer for assays
(Q-PCR, SDS-PAGE, and Western blotting, or arginase) or
replated at a concentration of 0.5 106 cells/ml in IMDM,
10% FCS, 150 mM MTG + growth factor used for their derivation (MCSF, GMCSF, IL-3) or treatment (MCSF + IL-4) during growth for stimulations (for NO assays or ELISAs).
10. To stimulate cells, replate in 6 wells (1 ml in a 6-well plate).
Add 10 ng/ml LPS to 3 wells and incubate at 37C, 5% CO2
for 24 h.
11. Harvest cell supernatants to an eppendorf tube and remove contaminating cells by microfuging at 13,000 g for 5 min. Divide
clarified supernatants into two fresh eppendorf tubes and store
at 20C until ready to assay supernatants (see Note 8).
3.2. Quantitative PCR
(see Note 9)
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Fig. 1. Q-PCR analysis of gene expression of argI and iNOS. Bone marrow aspirates were differentiated into macrophages
in the presence of 10 ng/ml of MCSF, GMCSF, or IL-3 for 10 days or in the presence of MCSF for 7 days followed by addition
of 10 ng/ml of IL-4 for an additional 3 days. After 10 days, macrophages were treated with 10 ng/ml of LPS for 24 h and
cells were harvested into TRIzol for RNA extraction. cDNA was prepared by reverse transcription and used for Q-PCR analysis. Expression of argI and iNOS were evaluated for each sample relative to b-glucuronidase (GUS) as an internal control.
Data shown are means SD for three independent experiments performed in triplicate.
4. Add APS (80 mL) and 20 mL TEMED and mix gently but
thoroughly by rocking the Falcon tube to avoid introducing air
bubbles. Pour the entire 40 ml solution between the glass plates.
Using a Pasteur pipet, gently add 5 ml of H2O-saturated butanol
to overlay the top of the gel. Be careful not to cause mixing with
the denser gel solution. Allow gel to polymerize about 30 min.
5. Pour off the alcohol overlay. Rinse the gel top with distilled
water and drain water well (see Note 17).
6. To make a 4% stacking gel, combine 9.75 ml water, 3.75 ml 4
stacking gel buffer, and 1.5 ml acrylamide stock solution. Add
75 ml APS and 15 ml TEMED in a 50 ml Falcon tube. Mix by
inversion and pipet onto the top of the separating gel. Place
the comb into the top of the gel. Avoid trapping air bubbles
below or on the side of the comb during insertion. Allow to
polymerize for 60 min before removing comb.
7. Using gel loading tips, add 100 ml of the sample (one-half) to
bottom of the wells.
8. Prepare 1.4 L of running buffer by diluting 140 ml of 10 running buffer stock solution to 1.4 L with dH2O. Gently add
running buffer to top up the wells with a Pasteur pipet and
then fill the upper buffer chamber with running buffer. Pour
the remaining running buffer into the bottom buffer reservoir
of the gel apparatus ensuring that it covers the bottom of the
gel and glass plates.
9. Fill the inner chamber of the gel apparatus with cold water and
run gel overnight (16 h) at 65 V.
3.4. Western Blotting
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GM
IL-3
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IL-4
Ym1
ArgI
GAPDH
Fig. 2. Western blot analysis of M2 macrophage marker protein expression. Bone marrow
aspirates were differentiated into macrophages in the presence of 10 ng/ml of MCSF,
GMCSF, or IL-3 for 10 days or in the presence of MCSF for 7 days followed by addition of
10 ng/ml of IL-4 for an additional 3 days. Macrophage cell lysates were separated on a
10% SDS-PAGE, transferred onto PVDF and probed for M2 macrophage markers, Ym1 and
argI, as well as GAPDH, as a loading control.
3.6. NO Assay
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Fig. 3. Analysis of enzymatic activity of argI and iNOS. Bone marrow aspirates were differentiated into macrophages in the
presence of 10 ng/ml of MCSF, GMCSF, or IL-3 for 10 days or in the presence of MCSF for 7 days followed by addition of
10 ng/ml of IL-4 for an additional 3 days. After 10 days, macrophages were harvested into arginase lysis buffer for arginase
activity assays or left untreated or treated with 10 ng/ml of LPS for 24 h. After LPS treatment, cell supernatants were
harvested, clarified and subjected to the Griess assay to measure NO2, downstream of NO production. Data shown are
means SD for three independent experiments performed in triplicate.
Fig. 4. Measurement of pro-inflammatory (IL-12 p40) and anti-inflammatory (IL-10) cytokine production and IL-12/IL-10
ratios. Bone marrow aspirates were differentiated into macrophages in the presence of 10 ng/ml of MCSF, GMCSF, or IL-3
for 10 days or in the presence of MCSF for 7 days followed by addition of 10 ng/ml of IL-4 for an additional 3 days.
Resulting macrophages were treated with LPS for 24 h and cell supernatants were harvested, clarified and assayed by
ELISA for IL-12p40 and IL-10 and the ratio of IL-12/IL-10 produced in response to LPS was calculated. Data shown are
means SD for four independent experiments assayed in duplicate.
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4. Notes
1. Nucleated cell counts can be performed by diluting cell
suspensions 1 in 20 in 3% acetic acid. This procedure lyses all
cells including red blood cells and the remaining nuclei can be
counted on a hemocytometer.
2. A thorough bone marrow flush results from two femurs and
two tibias gives up to 80 106 cells and approximately 90% of
cells remain in suspension after 4 h of adherence depletion.
The number of cells that become adherent does vary in some
genetically modified animals and so this is a very important
step if macrophages from genetically modified mice are being
compared to wild type mice.
3. It is much easier to resuspend cell pellets in a small volume of
medium (5 ml) and then to dilute it up to a larger volume.
This ensures a homogeneous cell suspension.
4. We have assayed several different sources of conditioned media
and the amount of growth factor that they provide varies
dramatically between source and batch. Because the procedure
described here aims to compare alternative activation strategies
based on growth factors used during differentiation, we recommend that recombinant sources of growth factor are used
to obtain similar results.
5. Cell concentration during derivation is important because
macrophage skewing during differentiation requires cell intrinsic and cell extrinsic factors. Cell extrinsic factors are affected
by cell concentration.
6. Macrophages derived in this way are consistently more than 95%
positive for Mac-1 and F4/80 by flow cytometric analysis.
7. Recombinant IL-13 also skews MCSF derived macrophages to
an alternatively activate phenotype, although it is less potent
than IL-4 when compared directly. IL-10 (10 ng/ml) enhances
IL-4 or IL-13 induced alternative activation of macrophages,
but it does not mediate skewing macrophages to an M2a phenotype on its own.
8. Cell supernatants harvested for ELISAs should be stored in aliquots because some cytokines are sensitive to freezethaw cycles.
9. RNA is extremely sensitive to degradation by ubiquitous
RNAses. All sample handling must be done with gloves, all
plasticware used should be RNAse free, and all water should be
treated with DEPC.
10. TRIzol containing samples should be handled in a fume hood.
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