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.
©

www.beckmancoulter.com/cell-research SEPTEMBER 2007 l CELLULAR RESEARCH 3

CellResearch.v3.final.qxd:Cellular Research 9/26/07 11:47 AM
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
system designed to provide reproducible data,
with a broad dynamic range, which represents
measured characteristics with a high degree of
accuracy. This can be achieved using high
resolution digital signal processing, which
converts analog inputs to digital values to obtain
high resolution analysis leading to accurate and
reproducible results.
In order to understand the measurement and
analysis component of an experiment, we need
to first understand the following characteristics
and how they affect flow cytometric data.
Definitions
Resolution:
The number of pixels in an image. The more
pixels, the higher the resolution. The higher the
resolution, the better the picture.
(www.americandynamics.net)
In relation to Flow Cytometry:
ADC (analog-to-digital converter) resolution
determines the accuracy of a measurement. The
higher the resolution (number of bits), the more
accurate the measurement. System resolution
describes an instrument’s ability to detect
differences in fluorescence intensity or size.
Accuracy:
The quality of being near to the true value.
(wordnet.princeton.edu)
In relation to Flow Cytometry:
The degree to which the measurement result
produced by the flow cytometer conforms to the
‘true’ value.1
Dynamic Range:
The range of input or output that a device can
process without overflow or distortion.
(dspvillage.ti.com)
In relation to Flow Cytometry:
The dynamic range of signal expression or particle
size in biological systems can be upwards of
10,000 times (4 log) between the lowest and
highest response.2
4 CELLULAR RESEARCH l SEPTEMBER 2007
Page 4
Figure 1. www.answers.com/topic/analog-to-digital-converter
Digital Signal Processing:
What is digital signal processing and why is it
important in the collection of my data?
Digital signal processing (DSP) is the conversion
of an analog signal—a voltage representing the
input signal—into a digital signal, i.e., a series of
integer values. This is performed by an analog-to-
digital converter (ADC). The resolution of the
converter refers to the number of discrete values it
can produce over the range of analog values.
The subsequent processing of signals is preferably
accomplished in the digital domain because it is
fast, accurate and reliable. Additionally, it allows
for further analysis of the data without loss of
resolution. DSP is also important in flow cytometric
acquisition because it obviates the need for
logarithmic amplifiers, which are notorious for
their non-linearity, and can introduce error in
measurement.
However, it is important to remember that not all
methods of DSP are the same. Two characteristics
can affect overall system performance.
1) When digitization occurs directly from the
amplifier, if one requires measurements
with <0.1% error, one needs to sample the
pulse 120 times in 3 µsecs, in other words,
by using at least a 40 MHz ADC.
2) Additionally, the resolution of the ADC affects
the usable dynamic range of the system, so
the higher the better.
In order to have good accuracy, which refers to
a usable dynamic range over 4 decades, at least
20-bit resolution is required. The Beckman Coulter
Flow Cytometers, including the XL and FC 500
Series, are equipped with revolutionary patented
DSP technology, providing high resolution 20-bit
data, employing elegant electronic switching
techniques designed to greatly improve the low
end resolution of the data.3
www.beckmancoulter.com/cell-research
 CellResearch.v3.final.qxd:Cellular Research
So how does this high resolution flow cytometry
provide accurate and reproducible results?
High resolution data translates into more
available channels to put the data into when
representing the actual input signal on the
histogram or dot plot. Sufficient ADC resolution
is important because of the wide dynamic range
of biological systems. Therefore, in order to see
the negative and positive events on the same
plot, the system requires at least 4 decades of
usable dynamic range. As mentioned, good
accuracy between the ADC output and log
channel display over 4 decades requires a
-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 2.
In the first decade, the channel width is wider
for all the signal traces because there are fewer
channels available. In this simplified example,
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
from all three signals coincide as the number of
channels is sufficient.
www.beckmancoulter.com/cell-research
9/26/07 11:47 AM Page 5
DIGITAL SIGNAL PROCESSING
What is the significance of the number and
width of the channels and how does it impact
the accuracy of my data and the dynamic
range?
Figure 3 illustrates the mapping of 20-bit and
18-bit data to a log scale. Each decade has an
increasing number of channels in which to place
data, with a maximum resolution of 1,048,575
channels for the 20-bit data. Notice that the
18-bit data, which has been converted to 18 bits
using a 14-bit ADC and a 16x multiplier, has a
maximum resolution of 262,144 channels5 ; ten
times less per decade than the 20-bit data.
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,
usable dynamic range for biological responses
of 4 decades. Figure 4B shows the accuracy for
a system that has 18-bit resolution. In this case,
the % Error* does not fall below 1% until the
Figure 4A. Figure 4B.
Figures 4A and 4B display the % Error* across the input
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.
SEPTEMBER 2007 l CELLULAR RESEARCH 5
CellResearch.v3.final.qxd:Cellular Research 9/26/07 11:47 AM
DIGITAL SIGNAL PROCESSING
second decade, leaving less than 3 decades of
usable dynamic range for the analysis of any type
of quantitative biological response. Note the
y-axis scales are adjusted to accommodate the
displayed values.
Let’s look at some data plots to see how
resolution and accuracy affect the data.
Figure 5A displays 20-bit data from 3 µm
fluorescent particles. The voltage has been ad-
justed to put these particles in each of the 4
decades. Notice the narrow peaks in all 4 plots
which exemplify the high accuracy shown across
all 4 decades. Alternatively, 18-bit data (Figure 5B)
collected using the same fluorescent particles
exhibits acceptable resolution in the upper
decades of the log display; however the increased
measurement error in the lower decade is
noticeable and may affect the data collected in
this area of the histogram.
Figure 5A.
Figure 5B.
6 CELLULAR RESEARCH l SEPTEMBER 2007
Page 6
So, high resolution digital data improves the
accuracy of my data and provides 4 decades
of usable dynamic range.
Yes, high resolution 20-bit digital data, available
on Beckman Coulter systems, produces results
with minimal error (<1% error) across the usable
4 decade dynamic range. Therefore, accurate
measurement of a biological response, that has
greater than a 3 log shift in expression, requires
a system which can provide sufficient resolution
over this dynamic range.
REFERENCES
1. Shapiro H. Practical Flow Cytometry, 4th Ed. Page 214.
2. Snow C. Cytometry Part A 2004 57A:63-69.
3. Auer B, et. al. Patent 5,367,474, issued November 1994.
4. Wood JCS. Resolution Requirements for the Digitization
of Flow Cytometry Data. 2006 Abstract 266, ISAC XXIII
International Conference.
5. BD FACSDiVa Option White Paper. 2002 Becton
Dickinson and Company.
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.

www.beckmancoulter.com/cell-research SEPTEMBER 2007 l CELLULAR RESEARCH 7

CellResearch.v3.final.qxd:Cellular Research 9/26/07 11:48 AM Page 8

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

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

CellResearch.v3.final.qxd:Cellular Research 9/26/07
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
using hemocytometry or automatic instrumen-
tation such as Vi-CELL. These methods use
Trypan Blue dye exclusion to distinguish viable
from non-viable cells under an optical
microscope or CCD camera. Flow cytometry
technology employs DNA-intercalating molecules
that fluoresce by interacting with a nucleic acid
basis. Distinction between viable and non-viable
cells results from the fact that the intact cell
membrane is impermeable to intercalating
molecules such as propidium iodide (PI) or
7-Amino-actinomycin D (7-AAD), whereas
compromised membrane is not, opening the
path to cellular DNA. The Quanta SC MPL, an
advanced, high throughput flow cytometer, uses
PI or 7-AAD to distinguish viable cells from non-
viable ones.
In this study, we compared cell count and
viability of Chinese Hamster Ovary (CHO) cells
in suspension culture determined by the Quanta
SC MPL with those obtained by the Vi-Cell XR.
It was also our intention to test the throughput,
efficiency, precision and accuracy of using
96-well plate format to measure viable cell
counts and viability using the Quanta SC MPL.
The auto sample preparation function, relatively
small sample volume (50-100 µL), and short
analysis time (~50 sec/sample) make the Quanta
SC MPL an ideal platform when high throughput
analysis is necessitated. In comparison, Vi-CELL
XR requires ~2.5 minutes to analyze a 500 µL
sample.
Materials and Methods
Cells. CHO cells in suspension culture were
used in the current study. Depending on cell
types and culture conditions, cell aggregates can
be minimized by additions of EDTA or Tween-20.
Vi-CELL XR Protocol. Standard CHO
parameters were used for analysis according
to the manufacturer’s instruction (Beckman
Coulter).
Quanta SC MPL Protocol. Cell culture samples
were mixed with EDTA at a final concentration of
10 mM, to minimize cell adhesion to the well
www.beckmancoulter.com/cell-research
11:48 AM Page 9
CELL COUNT & VIABILITY
surface, and pipetted into a 96-well poly-
carbonate plate (Axygen Scientific) at 100
!L/well. PI (Invitrogen) at a final concentration
of 2 !g/mL was added into each well by using
the automatic Well Prep function of the Quanta
SC MPL. The sample was mixed by redispensing
6 times at high speed before data acquisition.
Viability was assessed based on the PI
fluorescence signal on the FL3. Additional
instrument settings are summarized in Table 1.
Total Count 10,000
Run Volume 50
Peak Count 1,000,000
Time 0
Concentration Value Yes (Counting Time:
10 sec. Start Time: 5 sec.)
Table 1. Stop Sample Criteria.
Results
Cell counting linearity for suspension CHO cells
was established for the Quanta SC MPL through
a serial dilution experiment. A CHO cell sample
with ~90% viability and at a concentration of
~1.0 x 106 cells/mL was serially diluted with cell
culture medium from 0.06-1.00 x 106 cells/mL.
As shown in Figure 1A , the viable cell count of
the Quanta SC MPL correlates well with data
from Vi-Cell XR with a linear best fit slope of 1.07
and a R2 of 0.98. Results of cell viability are also
comparable between the Quanta SC MPL and
Vi-CELL XR (Figure 1B). Similar results were
obtained from CHO cells with ~70% or ~95%
cell viability (data not shown).
The Quanta SC MPL provides less viable cell
counts (Figure 2A) and viability (Figure 2B) than
the Vi-CELL XR when CHO cell viability is less
than ~50% measured by Vi-CELL XR. The
correlation of the viable cell count has a linear
best fit slope of 1.65 and an R2 of 0.98.
Viabilities measured by the Quanta SC MPL are
about 10% less than those obtained on the
Vi-CELL XR.
To determine if the Quanta SC MPL can be
employed for the 96-well high-throughput format
of cell counts and viability, a CHO cell sample
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
96-well polycarbonate plate. The same sample
was also repeatedly measured 96 times on a
Vi-CELL XR. Excellent correlations of viable cell
count and cell viability were obtained on both
systems (Figure 3, Tables 2 and 3).
count and viability were comparable between the
two instruments when cell viabilities were more
than ~70%, a range that was critical for cell
culture optimization in process development.
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

Figure 3. Viable cell counts and viability on the Quanta SC MPL
using the 96-well high-throuhput format and comparison to data
obtained from the Vi-Cell XR.
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
cells in suspension cultures. The present study
also demonstrates that, because of its automatic
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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