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CellResearch.v3.final.

qxd:Cellular Research 9/26/07 11:47 AM Page 1

ISSUE 3, SEPTEMBER 2007

Technology Issue

Digital Signal Processing


How Resolution, Accuracy
and Dynamic Range Affect
the “Final Answer”

Standardization Fundamentals
For Flow Cytometry
Measurements

Cell Count and Viability


High Throughput Analysis
by Quanta SC MPL

NL-10854A
CellResearch.v3.final.qxd:Cellular Research 9/26/07 11:47 AM Page 2

Solutions and News


Vi-CELL! XR Integrated for Fully Automated Cell Culture Application
Beckman Coulter recently announced the successful integration of its Vi-CELL XR Cell Viability Analyzer with the Biomek! Laboratory
Automation Workstation for real-time cellular imaging in a walk-away system for cell culture systems. Integrated automation adds systems
flexibility to the Vi-CELL instrument, allowing it to be used from basic cellular research through drug development to scale-up and even into
pharmaceutical manufacturing quality control. The Vi-CELL XR (extended range) measures the viability of cells and counts them in minutes,
using the widely accepted Trypan Blue cell viability tissue culture protocol. The instrument measures 15 to 30
times more volume than competitive systems in the same amount of time, with a more comprehensive
array of parameters. The automated, integrated solution monitors cell growth over time.
Beckman Coulter’s Integrated Solutions Group developed the customized solution for a cell
culture “seed and feed” application. Components of the solution include the Vi-CELL XR, a
Biomek NXP workstation configured with a Span-8 Pod and gripper, a Biomek FXP dual-bridge
workstation, storage devices, incubator, plate hotel and two detectors. The integrated solution
provides the cell analysis data necessary to evaluate clone selection, cell expansion and protein
expression results. Data from the analysis is also automatically captured for subsequent analysis
with other cellular results that determine cell viability and efficiency such as confluence, loci of
growth and protein-protein interactions.
“No other cell viability analyzer can be integrated for this level of automation,”
explained Karen Bezold, director of research flow cytometry and business
development for Beckman Coulter. “Because Beckman Coulter’s total product
offering is so broad, we are uniquely qualified to provide complete solutions
and support to our customers. This capability gives our users additional
downstream application options for their easy-to-use Vi-CELL
instruments.”
“Beckman Coulter’s Vi-CELL XR Cell Viability Analyzer with its
extended, 2–70 micron size range, reagent-saving 500 microliter sample
volume and enhanced image resolution can now be utilized in fully
automated cell culture systems with walk-away operation for the
monitoring and optimization of cell growth,” commented Bob Lund, group
manager, integrated solutions for Beckman Coulter. YS

Calendar of Events
SEPTEMBER
Jahrestagung der Deutschen Gesellschaft für Immunologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . September 5-8, Heidelberg, Germany
European Congress of Clinical Cell Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . September 6-8, Rotterdam, The Netherlands
Australasian Flow Cytometry Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . September 17-19, Melbourne, Australia
European Society for Domestic Animal Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . September 19-23, Celle, Germany
ASBMB ComBio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . September 22-26, Sydney, Australia
European Biotech Crossroads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . September 26-28, Lille, France
VHL Symposium: Cytometrie en Morfologie in de Dagelijkse Praktijk . . . . . . . . . . . . . . . . . . . . . September 27, Zwolle, The Netherlands
Great Lakes International Imaging and Flow Cytometry Association . . . . . . . . . . . . . . . . . . . . . September 28-30, Windsor, ON, Canada
IFCC / Beckman Coulter: Frontiers in Rare Event Analysis and Multiplex
Characterization of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . September 28-30, Bremen, Germany
OCTOBER
Club Hématopoïèse et Oncogénèse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . October 7-10, Giens, France
Clinical Cytometry Society . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . October 7-9, Washington, DC
American Society for Histocompatibility & Immunogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . October 8-12, Minneapolis, MN
Biotechnica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . October 9-11, Hannover, Germany
Association Française de Cytométrie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . October 10-12, Clermont-Ferrand, France
Jahrestagung der Deutschen Gesellschaft für Zytometrie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . October 10-13, Regensburg, Germany
World Congress on Regenerative Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . October 17-19, Leipzig, Germany
Turkish-US Cytometry Workshop . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . October 25-28, Istanbul, Turkey
NOVEMBER
Signal Transduction Society . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . November 1-3, Weimar, Germany
Japanese Society of Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . November 20-22,Tokyo, Japan
Workshop Stammzelltransplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . November 22-23, Frankfurt / Main, Germany
Société Française d'Immunologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . November 26-29, Lyon, France
Swiss Cytometry Society . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . November 29-30, Bern, Switzerland
Association Française des Sciences et Techniques de l'Animal de Laboratoire . . . . . . . . . . . November 28-30, Reims, France
DECEMBER
Australasian Society of Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . December 2-6, Sydney, Australia
American Society of Hematology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . December 8-11, Atlanta, GA
Biochemistry and Molecular Biology 2007 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . December 11-15, Yokohama, Japan
Indo-US Cytometry Workshop . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . December 17-22, Lucknow, India
Annual Meeting, Dutch Society for Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . December 20-21, Noordwijkerhout,
The Netherlands
CellResearch.v3.final.qxd:Cellular Research 9/26/07 11:47 AM Page 3

Inside
2 Solutions and News
Calendar of Events
4 Improved Accuracy and Dynamic
Range through Patented Digital
Signal Processing
7 Fundamentals of Standardization
for Flow Cytometric Measurements
9 High throughput Analysis of Cell Count
Cellular Research is published by and Viability by Quanta™ SC MPL:
Beckman Coulter, Inc. for our customers. A Comparison Study of Data Obtained
Our mission is to provide research from Vi-CELL! XR
professionals with current
industry information and solutions. 11 Quanta Systems Resource Guide
We value your feedback.
To submit articles, suggestions, or

Letter from the Editor


to be added to the mailing list, please
contact us at:
By mail:
When it comes to meaningful—high content, high throughput—investigations in cellular
Beckman Coulter, Inc.
analysis, flow cytometry is the technology; providing an astounding amount of relevant
Attn.: Cellular Research Editor
200 S. Kraemer Blvd. m/s W-362 information in a relatively short period of time.
P.O. Box 8000 Through nanometric optical means, multiparametric flow cytometers discriminate
Brea, CA 92822-8000 U.S.A. cells on the basis of surface and intra-cellular labeled molecules, in addition to light scattering-
associated relative size and shape properties. Through sophisticated fluidic means, it does this
By e-mail:
with precision and at an amazing rate of tens of thousands per second. Thus, nearly all
www.beckman.com/cell-research
scientists recognize that flow cytometry has seized an eminent position of powerful, flexible,
editors: as well as highly accurate technology.
Michel Herbert In this issue of Cellular Research we will get down to the heart of the flow cytometric
Laura Nichelson business; the hardware. To illustrate the power of this technology we present various good
practices that are easily achieved with Beckman Coulter’s Quanta™ SC and FC 500 series
contributors: flow cytometers.
Lori Charie The Quanta SC is capable of measuring cell density and viability in a high throughput
Louis Cheung fashion with the MPL 96-well plate option, streamlining labwork and raising confidence in the
Diane Lary results at the same time.
Shun Luo Standardization is a key process required to answer the question of how a given system
Jorge Quintana
is performing at any point of time and how results compare across various system platforms.
Yong Song
It is one of the fundamentals of Beckman Coulter Flow Cytometry and we are dedicated to
design: bringing efficient standardization tools in hardware design as well as in the accompanying
Teri Beauchamp reagent portfolio, ultimately leading users to clear data interpretation.
The dynamic range of an analytical system, as demanded by flow cytometry, has to be
addressed with speed as well as accuracy. At Beckman Coulter, the patented hardware and
basic tools used to support such a dynamic range has a 20-bit capacity, resolving the dimmest
Eastern Europe, Middle East, North Africa, particles as comfortably as the brightest ones from true negative events. Our unique patent
South West Asia: Switzerland, Nyon (41) 22 365 3707. uses only 1 ADC (analog-to-digital converter) per channel and adjusts the signal based on the
Australia, Gladesville (61) 2 9844 6000.
Canada, Mississauga (1) 905 819 1234. voltage input. Common flow cytometric data produced by many competitive systems are still
China, Beijing (86) 10 6515 6028. only 14-16 bit-powered.
Czech Republic, Prague (420) 1267 00 83 66.
Hong Kong (852) 2814 7431, 2814 0481.
When choosing a flow cytometer, we believe the key features described in this issue will
France, Villepinte (33) 1 49 90 90 00. help you determine how the instruments you’re reviewing convert the cellular characteristics
Germany, Krefeld (49) 2151 33 35. into dependable electronic signals, and how these signals are then standardized, processed
India, Mumbai (91) 22 3080 5101.
Italy, Cassina de’ Pecchi (Milan) (39) 02 953921. and measured.
Japan, Tokyo (81) 3 6745 4704.
Latin America (1) (305) 380 4709. Cordially,
Mexico, Mexico City (52) 55 560 57770.
Netherlands, Mijdrecht (31) 297 230630.
Puerto Rico (787) 747 3335. Singapore (65) 6339 3633.
South Africa/Sub-Saharan Africa,
Johannesburg (27) 11 805 2014.
Sweden, Bromma (46) 8 564 85 900. Michel Herbert
Switzerland, Nyon 0800 850 810. Editor
Taiwan, Taipei (886) 2 2378 3456.
Turkey, Istanbul (90) 216 309 1900.
Cellular Analysis, Beckman Coulter, Inc.
UK, High Wycombe (44) 01494 441181.
USA, Brea, CA (1) 800 352 3433, (1) 714 993 5321.
B2007-8003 50K 2007 Beckman Coulter, Inc. PRINTED IN U.S.A.
©

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CellResearch.v3.final.qxd:Cellular Research 9/26/07 11:47 AM Page 4

DIGITAL SIGNAL PROCESSING

Improved Accuracy and Dynamic Range


through Patented Digital Signal Processing
By Diane Lary, Cellular Analysis Business Center, Beckman Coulter, Miami, FL

How Resolution, Accuracy and Dynamic Range


of the Cytometer Affect the ‘Final Answer’

With all the variables involved in planning a


biological experiment, if an assay requires flow
cytometric analysis as the measurement com-
ponent, one prefers a high-resolution analytical
Figure 1. www.answers.com/topic/analog-to-digital-converter
system designed to provide reproducible data,
with a broad dynamic range, which represents Digital Signal Processing:
measured characteristics with a high degree of
What is digital signal processing and why is it
accuracy. This can be achieved using high
important in the collection of my data?
resolution digital signal processing, which
converts analog inputs to digital values to obtain Digital signal processing (DSP) is the conversion
high resolution analysis leading to accurate and of an analog signal—a voltage representing the
reproducible results. input signal—into a digital signal, i.e., a series of
integer values. This is performed by an analog-to-
In order to understand the measurement and
digital converter (ADC). The resolution of the
analysis component of an experiment, we need
converter refers to the number of discrete values it
to first understand the following characteristics
can produce over the range of analog values.
and how they affect flow cytometric data.
The subsequent processing of signals is preferably
Definitions
accomplished in the digital domain because it is
Resolution: fast, accurate and reliable. Additionally, it allows
The number of pixels in an image. The more for further analysis of the data without loss of
pixels, the higher the resolution. The higher the resolution. DSP is also important in flow cytometric
resolution, the better the picture. acquisition because it obviates the need for
(www.americandynamics.net) logarithmic amplifiers, which are notorious for
In relation to Flow Cytometry: their non-linearity, and can introduce error in
ADC (analog-to-digital converter) resolution measurement.
determines the accuracy of a measurement. The
However, it is important to remember that not all
higher the resolution (number of bits), the more
methods of DSP are the same. Two characteristics
accurate the measurement. System resolution
can affect overall system performance.
describes an instrument’s ability to detect
differences in fluorescence intensity or size. 1) When digitization occurs directly from the
amplifier, if one requires measurements
Accuracy:
with <0.1% error, one needs to sample the
The quality of being near to the true value.
pulse 120 times in 3 µsecs, in other words,
(wordnet.princeton.edu)
by using at least a 40 MHz ADC.
In relation to Flow Cytometry:
The degree to which the measurement result 2) Additionally, the resolution of the ADC affects
produced by the flow cytometer conforms to the the usable dynamic range of the system, so
‘true’ value.1 the higher the better.
Dynamic Range: In order to have good accuracy, which refers to
The range of input or output that a device can a usable dynamic range over 4 decades, at least
process without overflow or distortion. 20-bit resolution is required. The Beckman Coulter
(dspvillage.ti.com) Flow Cytometers, including the XL and FC 500
In relation to Flow Cytometry: Series, are equipped with revolutionary patented
The dynamic range of signal expression or particle DSP technology, providing high resolution 20-bit
size in biological systems can be upwards of data, employing elegant electronic switching
10,000 times (4 log) between the lowest and techniques designed to greatly improve the low
highest response.2 end resolution of the data.3

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DIGITAL SIGNAL PROCESSING

So how does this high resolution flow cytometry What is the significance of the number and
provide accurate and reproducible results? width of the channels and how does it impact
High resolution data translates into more the accuracy of my data and the dynamic
available channels to put the data into when range?
representing the actual input signal on the Figure 3 illustrates the mapping of 20-bit and
histogram or dot plot. Sufficient ADC resolution 18-bit data to a log scale. Each decade has an
is important because of the wide dynamic range increasing number of channels in which to place
of biological systems. Therefore, in order to see data, with a maximum resolution of 1,048,575
the negative and positive events on the same channels for the 20-bit data. Notice that the
plot, the system requires at least 4 decades of 18-bit data, which has been converted to 18 bits
usable dynamic range. As mentioned, good using a 14-bit ADC and a 16x multiplier, has a
accuracy between the ADC output and log maximum resolution of 262,144 channels5 ; ten
channel display over 4 decades requires a times less per decade than the 20-bit data.
-to-digital-converter
system with at least 20-bit resolution. On a
system with lower resolution there are fewer
numbers of channels to distribute the data into.
As such, each channel represents a larger
change in input signal, making it difficult to
resolve small changes in fluorescence intensity.
This is easier to see in the lower decades of a
log signal display. Figure 2 shows an example of
data with different bit resolution and graphically
exhibits what impact the resolution has on the
display of the data.4 Figure 3.
Fewer channels in the lower decades, combined
with intrinsic background noise, make it more
difficult to accurately measure small changes in
signal or fluorescence intensity.
On a system which provides 20-bit resolution,
one is able to obtain very accurate data (<1%
Error*) across the complete dynamic range.
Figure 4A illustrates superior accuracy across
all decades on the log histogram of the Beckman
Coulter FC500 cytometer. It uses the patented
20-bit DSP technology, providing a quantitative,
Figure 2.
usable dynamic range for biological responses
In the first decade, the channel width is wider of 4 decades. Figure 4B shows the accuracy for
for all the signal traces because there are fewer a system that has 18-bit resolution. In this case,
channels available. In this simplified example, the % Error* does not fall below 1% until the
the 10-bit trace only displays 2 channels
in the first decade, the 14-bit trace appears
choppy, which is commonly represented as
“Picket Fencing” in some log displays. However,
the 20-bit signal has sufficient channels to
display highly accurate data in this decade. In
decades 2 & 3, the data smoothes out somewhat
because there are more channels available to
distribute data into. In the 4th decade, the data Figure 4A. Figure 4B.
from all three signals coincide as the number of Figures 4A and 4B display the % Error* across the input
channels is sufficient. voltage range of 0—10 volts. The vertical dotted red line on the
20-bit display represents the crossover point used in the
Beckman Coulter patented electronic switching method which
improves the accuracy of the data in the lower end of the log
display. The horizontal dotted red line on the 18-bit display
represents the 5% Error mark.
* % Error based on simulated best case data with zero noise and zero offset.

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DIGITAL SIGNAL PROCESSING

second decade, leaving less than 3 decades of So, high resolution digital data improves the
usable dynamic range for the analysis of any type accuracy of my data and provides 4 decades
of quantitative biological response. Note the of usable dynamic range.
y-axis scales are adjusted to accommodate the Yes, high resolution 20-bit digital data, available
displayed values. on Beckman Coulter systems, produces results
Let’s look at some data plots to see how with minimal error (<1% error) across the usable
resolution and accuracy affect the data. 4 decade dynamic range. Therefore, accurate
Figure 5A displays 20-bit data from 3 µm measurement of a biological response, that has
fluorescent particles. The voltage has been ad- greater than a 3 log shift in expression, requires
justed to put these particles in each of the 4 a system which can provide sufficient resolution
decades. Notice the narrow peaks in all 4 plots over this dynamic range.
which exemplify the high accuracy shown across REFERENCES
all 4 decades. Alternatively, 18-bit data (Figure 5B) 1. Shapiro H. Practical Flow Cytometry, 4th Ed. Page 214.
collected using the same fluorescent particles 2. Snow C. Cytometry Part A 2004 57A:63-69.
exhibits acceptable resolution in the upper 3. Auer B, et. al. Patent 5,367,474, issued November 1994.
decades of the log display; however the increased 4. Wood JCS. Resolution Requirements for the Digitization
measurement error in the lower decade is of Flow Cytometry Data. 2006 Abstract 266, ISAC XXIII
noticeable and may affect the data collected in International Conference.
this area of the histogram. 5. BD FACSDiVa Option White Paper. 2002 Becton
Dickinson and Company.

Figure 5A.

Figure 5B.

6 CELLULAR RESEARCH l SEPTEMBER 2007 www.beckmancoulter.com/cell-research


CellResearch.v3.final.qxd:Cellular Research 9/26/07 11:47 AM Page 7

STANDARDIZATION

Fundamentals of Standardization for Flow


Cytometric Measurements
By Lori A. Charie, Jorge Quintana, Cellular Analysis Business Center, Beckman Coulter, Inc., Miami FL
To ensure scientifically sound and longitudinally
comparable results there is a critical need to
characterize flow cytometric measurements
utilizing a standardized approach that will
adequately address the key performance
characteristics of each flow cytometer.
Instrument performance may be established by
addressing three areas: precision (the repro-
ducibility of measurements of identical particles),
linearity and sensitivity. This can be performed by
using brightly fluorescent beads of uniform size.
Precision may be verified through an instrument Figure 1: Flow-Check™ Fluorospheres run on a
alignment / fluidics check, ensuring that the Beckman Coulter, Inc. FC 500 Flow Cytometer.
sheath fluid is moving through the flow cell freely
and without turbulence that could be an manufacturer and should be monitored over time
indication of protein residue in the flow cell. A to detect trends (see Figure 1).
properly cleaned and aligned instrument will
produce symmetrical peaks with low variance; Linearity
this is particularly critical for DNA measurements. The linearity (or nonlinearity) of a flow cytometer
provides information about the accuracy of the
Linearity is fundamental to compensation
system, specifically the response of the
(correction for spectral overlap) and quantitative
electronics throughout the dynamic voltage range
measurements and may be assessed by using a
to be used. For this reason, it is critical to ensure
combination of two beads of different fluorescent
a linear response in order to accurately perform
intensities.
color compensation. The linearity of the PMT
Sensitivity is a measure of the ability of the response can be established using pairs of
system to resolve a dim fluorescent signal from fluorescent beads, such as IMMUNO-BRITE™
background and to resolve two dim populations. Fluorospheres for FL1-FL4 and Flow-Check™
A combination of two dim and one bright 770 and Flow-Set™ 770 for FL5. The ratio of
fluorescent bead with similar intrinsic CVs that the fluorescent intensities of a pair of beads is
have been calibrated to the number of equiv- calculated and plotted against the increasing
alent soluble fluorophores is used to quantitate voltage. The high voltage selected for any
sensitivity. application should be on the linear section of the
By combining these methods into an automated resulting curve (see Figures 2 and 3).
standardization routine, the means to uniquely
characterize and monitor each flow cytometry
system may be achieved. Beckman Coulter
introduced the concept of automated Flow
Cytometer standardization in the early 1990s and
incorporates, in its FC 500 Series, a complete
solution for flow cytometer standardization.

Essential Characterizations
Precision (Alignment / Fluidics)
Precision (instrument alignment and fluidics
performance) may be determined by running
brightly fluorescent beads such as Flow-Check™
Fluorospheres. Brightly fluorescent beads
ensure that photon statistics do not make a Figure 2: “Bead March” (using FL1 as an example) produced by
significant contribution to fluorescence gradual increasing voltages (A to E histograms from 1st to 5th charts)
variability1,5. The HPCV (Half peak CV) should be over the dynamic range of the instrument using IMMUNO-BRITE™
within the range stated by the instrument Fluorospheres.

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STANDARDIZATION
Conclusion
Practical Standardization Program
Establishing and maintaining a flow cytometer in a
way to assure optimal system performance is
based on the three fundamental characteristics of
precision, accuracy, and sensitivity. These can be
addressed in a laboratory standardization program
by following these steps:
Figure 3. Ratio of fluorescence intensities of the two beads plotted against voltage
(left chart) and the %CV plotted against voltage (right chart). The PMT begins to
1. Precision: monitor HPCVs of brightly
demonstrate a nonlinear response at the inflection point on either chart. In the case fluorescent beads daily.
of the example instrument, this inflection point is at around 400 V for all PMTs. 2. Accuracy: establish the PMT response when
the instrument is received and after
Sensitivity replacement of a PMT or major changes to
Characterization of the sensitivity of a flow the instrument.
cytometry system addresses the question of how 3. Sensitivity: determine the sensitivity of an
well an instrument is able to resolve dim instrument monthly and after any major
populations from each other and from modifications to the instrument.
background. The measure of molecules of
equivalent soluble fluorophore (MESF) provides
a means of translating sensitivity into a directly
measurable unit. Sensitivity can be described in
terms of two factors – the detection efficiency
(Q) and the background light level (B). The
detection efficiency is the number of
photoelectrons produced per molecule of
fluorochrome. Since both Q and B influence the
spread or standard deviation of the fluorescence
peak, analysis of Q and B provide a predictive
method for estimating the overlap, and therefore
resolution, between fluorescent peaks. Q and B
values are determined empirically by using a set
of at least two dim beads and a bright fluor-
escence calibrator bead. The CVs of the dim
beads are measured using the linear scale for
fluorescence intensity. The fluorescence of the
dim beads is converted from intensity units to Figure 4: Sensitivity threshold determination using Spherotech
MESF using the calibrator bead and the CVs of SPHERO™ Rainbow Calibration Particles.
the dim beads are then converted to SD2 using
the formula :
1
REFERENCES
SD2 = (MESF)2 x (CVdim2 – CVbright2) 1. Hoffman RA, Wood JCS. Current Protocols in Cytometry.
New York: John Wiley & Sons, Inc.; 2007.
A linear regression is performed correlating the 2. Wood JCS. Fundamental flow cytometer properties
SD2 (y-values) to the MESF (x-values) to obtain governing sensitivity and resolution. Cytometry 1998
the detection efficiency (Q) and the background 33:260-266.
light level (B). 3. Chase ES, Hoffman RA. Resolution of dimly fluorescent
particles: a practical measure of fluorescence sensitivity.
Once the Q and B values – conveniently Cytometry 1998 33:267-279.
calculated using a dedicated spreadsheet – 4. Schwartz A, Fernández-Repollet E, Vogt R, Gratama JW.
that represent adequate resolution for the system Standardizing flow cytometry: construction of a
have been established, they may be monitored standardized fluorescence calibration plot using matching
over time using a Levey-Jennings plot to ensure spectral calibrators. Cytometry 1996 26:22-31.
system performance. 5. Shapiro HM. Practical Flow Cytometry Fourth Edition.
New York: Wiley-Liss; 2003.
A typical presentation of an alternative method,
which measures threshold but not resolution, is To read complete article please go to
depicted in Figure 4. www.beckmancoulter.com/cell-lab

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CellResearch.v3.final.qxd:Cellular Research 9/26/07 11:48 AM Page 9

CELL COUNT & VIABILITY

High throughput Analysis of Cell Count and Viability by


Quanta™ SC MPL: A Comparison Study of Data Obtained
from Vi-CELL! XR
By Louis Cheung1, Yong Song2 and Shun Luo1,
1
Late Stage Cell Culture, Process Research & Development, Genentech, Inc., South San Francisco, CA 94080
2
Cellular Analysis Business Group, Beckman Coulter, Inc. Fullerton, CA 92835

Cell count and viability are commonly determined surface, and pipetted into a 96-well poly-
using hemocytometry or automatic instrumen- carbonate plate (Axygen Scientific) at 100
tation such as Vi-CELL. These methods use !L/well. PI (Invitrogen) at a final concentration
Trypan Blue dye exclusion to distinguish viable of 2 !g/mL was added into each well by using
from non-viable cells under an optical the automatic Well Prep function of the Quanta
microscope or CCD camera. Flow cytometry SC MPL. The sample was mixed by redispensing
technology employs DNA-intercalating molecules 6 times at high speed before data acquisition.
that fluoresce by interacting with a nucleic acid Viability was assessed based on the PI
basis. Distinction between viable and non-viable fluorescence signal on the FL3. Additional
cells results from the fact that the intact cell instrument settings are summarized in Table 1.
membrane is impermeable to intercalating
Total Count 10,000
molecules such as propidium iodide (PI) or
7-Amino-actinomycin D (7-AAD), whereas Run Volume 50
compromised membrane is not, opening the Peak Count 1,000,000
path to cellular DNA. The Quanta SC MPL, an Time 0
advanced, high throughput flow cytometer, uses Concentration Value Yes (Counting Time:
PI or 7-AAD to distinguish viable cells from non- 10 sec. Start Time: 5 sec.)
viable ones. Table 1. Stop Sample Criteria.

In this study, we compared cell count and Results


viability of Chinese Hamster Ovary (CHO) cells Cell counting linearity for suspension CHO cells
in suspension culture determined by the Quanta was established for the Quanta SC MPL through
SC MPL with those obtained by the Vi-Cell XR. a serial dilution experiment. A CHO cell sample
It was also our intention to test the throughput, with ~90% viability and at a concentration of
efficiency, precision and accuracy of using ~1.0 x 106 cells/mL was serially diluted with cell
96-well plate format to measure viable cell culture medium from 0.06-1.00 x 106 cells/mL.
counts and viability using the Quanta SC MPL. As shown in Figure 1A , the viable cell count of
The auto sample preparation function, relatively the Quanta SC MPL correlates well with data
small sample volume (50-100 µL), and short from Vi-Cell XR with a linear best fit slope of 1.07
analysis time (~50 sec/sample) make the Quanta and a R2 of 0.98. Results of cell viability are also
SC MPL an ideal platform when high throughput comparable between the Quanta SC MPL and
analysis is necessitated. In comparison, Vi-CELL Vi-CELL XR (Figure 1B). Similar results were
XR requires ~2.5 minutes to analyze a 500 µL obtained from CHO cells with ~70% or ~95%
sample. cell viability (data not shown).
Materials and Methods The Quanta SC MPL provides less viable cell
Cells. CHO cells in suspension culture were counts (Figure 2A) and viability (Figure 2B) than
used in the current study. Depending on cell the Vi-CELL XR when CHO cell viability is less
types and culture conditions, cell aggregates can than ~50% measured by Vi-CELL XR. The
be minimized by additions of EDTA or Tween-20. correlation of the viable cell count has a linear
best fit slope of 1.65 and an R2 of 0.98.
Vi-CELL XR Protocol. Standard CHO
Viabilities measured by the Quanta SC MPL are
parameters were used for analysis according
to the manufacturer’s instruction (Beckman about 10% less than those obtained on the
Coulter). Vi-CELL XR.

Quanta SC MPL Protocol. Cell culture samples To determine if the Quanta SC MPL can be
were mixed with EDTA at a final concentration of employed for the 96-well high-throughput format
10 mM, to minimize cell adhesion to the well of cell counts and viability, a CHO cell sample

www.beckmancoulter.com/cell-research SEPTEMBER 2007 l CELLULAR RESEARCH 9


CellResearch.v3.final.qxd:Cellular Research 9/26/07 11:48 AM Page 10

CELL COUNT & VIABILITY

Figure 1. Linearity and correlation of viable cell count (A) and viability (B; n = 4) using
the Quanta SC MPL and the Vi-Cell XR with a CHO cell sample with 90% viability.

Figure 2. Linearity and correlation of viable cell count (A) and viability (B) using the
Quanta SC MPL and the Vi-Cell XR with a CHO cell sample with ~50% viability.

with ~70% viability and at a concentration of Discussion and Conclusion


~0.35 x 10 cells/mL was mixed with EDTA at a
6 In the current study, we found the readings in cell
final concentration of 10 mM and aliquoted into a count and viability were comparable between the
96-well polycarbonate plate. The same sample two instruments when cell viabilities were more
was also repeatedly measured 96 times on a than ~70%, a range that was critical for cell
Vi-CELL XR. Excellent correlations of viable cell culture optimization in process development.
count and cell viability were obtained on both
systems (Figure 3, Tables 2 and 3). For samples with less than ~50% cell viability,
the viable cell count and cell viability obtained
from the Quanta SC MPL were consistently
less (10-15% lower) than those measured by the
Vi-CELL XR. This discrepancy is in agreement
with former studies (Altman, et al., 1993) and is
mainly due to differences in permeability of PI
and Trypan Blue to various degrees of cell
plasma membrane damages.

The Quanta SC MPL successfully demonstrated


the capability of measuring viable cell density
and viability in a high throughput fashion with the
96-well plate. It takes approximately 50 seconds
to process one sample on the Quanta SC MPL.
It is thus rational to design assays that will be
able to distinguish minor differences in viability of
Figure 3. Viable cell counts and viability on the Quanta SC MPL cells in suspension cultures. The present study
using the 96-well high-throuhput format and comparison to data also demonstrates that, because of its automatic
obtained from the Vi-Cell XR. sample preparation and plate monitoring
functions, database management and speed of
batch-based data analysis, the Quanta SC MPL
can be used in a much higher throughput fashion

10 CELLULAR RESEARCH l SEPTEMBER 2007 www.beckmancoulter.com/cell-research


CellResearch.v3.final.qxd:Cellular Research 9/26/07 11:48 AM Page 11

CELL COUNT & VIABILITY

Quanta SC MPL Vi-Cell XR Quanta SC MPL Vi-Cell XR


Mean* (x 106 cells/mL) 0.31 0.36 Mean 67.8% 68.1%
Standard Deviation 0.04 0.03 Standard Deviation 1.2 2.3
CV 12.3% 9.3% CV 1.8% 3.4%
*No statistically significant difference (student’s t-test: p > 0.05, n=96) Table 3. Mean, standard deviation and %CV on viability measurement.
Table 2. Mean, standard deviation and %CV on viable cell
density measurement.

and potentially enables “walk-away” automation REFERENCE


for cell count and viability measurement for in- 1. Altman SA, Randers A, Rao G. Comparison of
dustrial scale cell culture process development. Trypan Blue Dye Exclusion and Fluorometric
Using this high through-put flow cytometry Assays for Mammalian Cell Viability Determinations.
technology, we also expect that the Quanta SC Biotechnology Progress 1993 9: 871-874.
MPL will provide extended capability to carry out
multiplex assays of CHO cells for apoptosis
analysis, cell cycle profiling, mitochondria potential
determination and dehydrogenase activity.

QUANTA™ RESOURCE GUIDE


Selected Peer-reviewed Publications with Data Obtained from
Quanta Flow Cytometry Systems
ˆ
Rudolf E, Rudolf K, Cervinka M. Selenium activates p53 and p38 pathways and induces caspase-independent cell death in cervical cancer cells.
Cell Biol Toxicol. Online Pub DOI 10 1007/s10565-007-9022-1. Application: Annexin V-FITC/propidium iodide apoptosis analysis, phospho-p53,
phospho-JNK, phospho-p38, and Bax determination.
Sharma A, Comstock CES, Knudsen ES, Cao KH, Hess-Wilson JK, Morey LM, Barrera J, Knudsen KE. Retinoblastoma Tumor Suppressor Status
Is a Critical Determinant of Therapeutic Response in Prostate Cancer Cells. Cancer Res. 2007 67: 6192-6203. Application: Cell cycle analysis with
propidium iodide (PI) staining. Bromodeoxyuridine (BrdUrd) incorporation.
Lee HT, Kim M, Kim J, Kim N, Emala CW. TGF-Beta1 Release by Volatile Anesthetics Mediates Protection against Renal Proximal Tubule Cell
Necrosis. Am J Nephrol 2007 27: 416–424. Application: Annexin V-FITC/propidium iodide apoptosis analysis.
Li Q, Gerena L, Xie L, Zhang J, Kyle D, Milhous W. Development and validation of flow cytometric measurement for parasitemia in cultures of
P. falciparum vitally stained with YOYO-1. Cytometry Part A 2007 71A:297-307. Application: Fast, highly sensitive, reproducible and accurate
measurement for parasitaemia.
Urwin NAR, Horsnell J, Moon T. Generation and characterisation of colchicine-induced autotetraploid Lavandula angustifolia. Euphytica 2007
156:257–266. Application: Absolute genome size or C-values determination with PI staining.
Miyazaki H, Shiozaki A, Niisato N, Marunaka Y. Physiological significance of hypotonicity-induced regulatory volume decrease: reduction in
intracellular Cl– concentration acting as an intracellular signaling. Am J Physiol Renal Physiol 2007 292: F1411-F1417.
Applications: Accurate cell size measurement by Electronic Volume (EV). Intracellular Cl-concentration ([Cl-]i) measurement by a halide-specific fluorescent
dye, N-(6-methoxyquinolyl) acetoethyl ester (MQAE). Note: It is the first publication to demonstrate significance of normalizing fluorescence with cell volume
(EV), i.e. fluorescence concentration (FC), in measuring [Cl-]i.
Krishan A, Ganjei-Azar P, Jorda M, Hamelik RM, Reis IM, Nadji M. Detection of tumor cells in body cavity fluids by flow cytometric and
immunocytochemical analysis. Diag Cytopathol 2006 34:528-541. Application: High resolution DNA and nuclear volume measurement.
Bauer S, Yu LK, Demetri GD, Fletcher JA. Heat shock protein 90 inhibition in imatinib-resistant gastrointestinal stromal tumor. Cancer Res
2006 66: 9153-9161. Application: Cell cycle analysis with NIM-DAPI.
Cabana R, Frolova EG, Kapoor V, Thomas RA, Krishan A, Telford WG. The minimal instrumentation requirements for Hoechst side population
analysis: Stem cell analysis on low-cost flow cytometry platforms. Stem Cells 2006 24:2573-2581. Application: Stem cell side population analysis.
Weinberger LS, Burnett JC, Toettcher JE, Arkin AP, Schaffer DV. Stochastic gene expression in a lentiviral positive-feedback loop:
HIV-1 Tat fluctuations drive phenotypic diversity. Cell 2005 122:169-182 (data in Supplemental Data section). Application: Detection of
GFP expression. Cell size measurement by EV. Cell cycle analysis with NIM-DAPI. Cell cycle analysis and nuclei size measurement on GFP expressed cells.
Note: The paper described a very interesting phenomenon. After NIM (a solution contained NP-40) treatment, 2% formaldehyde fixed GFP expressed Jurkat
cells display GFP fluorescence equivalent to unfixed, non-NIM-treated cells. However, the fixed and NIM-treated cells have the same volume measurement as
bare nuclei (~5 µm diameter) because formaldehyde fixation plus NIM-lysing still permits ions to infuse the fixed cytoplasm but not the nucleus. Therefore,
fixed GFP expressed cell with NIM treatment maintains a halo of GFP around the major volume determinant, i.e. nucleus. This observation allowed the authors
to design the experiment to determine correlation of aneuploidy with GFP expression. YS
For a complete list of publications, visit www.beckmancoulter.com/cell-research

www.beckmancoulter.com/cell-research SEPTEMBER 2007 l CELLULAR RESEARCH 11


CellResearch.v3.final.qxd:Cellular Research 9/26/07 11:48 AM Page 12

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