Validaon of direct volume cell concentraon measure- ments using counng beads. Viable cell concentraon measurements are similar using direct volume, counng bead or hemacytometer methods. Platelet concentraons in human blood samples are obtained using fluorescence triggering on a flow cytometer. The direct volume method is more precise for measuring cell concentraon than counng bead or hemacytometer methods. Peristalc pumps allow direct cell concentraon measurements and high data acquision rates. Development of a unique peristalc pump-driven laminar flow fluidics system combines the advantages of hydro- dynamically focused cell sampling (high acquision rates, good light scaer and fluorescence resoluon) with the ability to report absolute cell counts for any idenfied populaon in a sample. Comparison of cell concentraon measurements by two methods. The x axis represents cell concentraon calculated by absolute cell counts in relaon to volume sampled. The y axis represents cell concentraon as calculated by the absolute cell counts in re- laon to the number of SPHERO TM AccuCount beads measured. 75 different samples were analyzed in five independently run experi- ments. Cell types analyzed included human peripheral blood sam- ples and mammalian cell lines. The average coefficient of variaon for replicate cell counts using three different counng methods on the same samples. A paired student’s T test was used to determine p values (95% confidence, N = 23). The direct-volume measurement method provides the least variability between replicate cell counts (average CV values: direct-volume measurement =2.10%; counng bead method =2.94%; hemacytometer =19.51%). Aliquots of whole blood were diluted 1:10 into HEPES-buffered saline with 1% formaldehyde. 20 µL aliquots of diluted blood were incubated with 20 µl CD41-PE anbody (DAKO clone 5B12) for 20 minutes in the dark at room temperature. Samples were then diluted with 1 mL HEPES-buffered saline with 1% formaldehyde. 5.0 µL of RFP-50-5 beads (Spherotech) were added to allow comparison of two counng methods. Triggering data collecon on forward scaer. Same sample triggered on fluorescence, showing improved discriminaon of platelets. Comparison of platelet counts obtained for two methods of counng. All data was collected using CD41-PE FL2 Trigger. Program #2785 Results Conclusions Technology giving researchers the ability to quickly and precisely deter- mine the absolute number or concentraon of cells with a given pheno- type is of great interest in diverse fields including clinical research and diagnoscs, drug discovery, and cell biology. The recent advent of small, fully digital flow cytometers combines the advantages of hydrodynami- cally focused cell sampling, high data acquision rates and excellent light scaer resoluon with the ability to calculate absolute counts for any idenfied populaon in a sample. In the study presented here, sev- eral different cell counng applicaons are explored: viability determi- naon for cultured cell lines, platelet counts in whole, unlysed human peripheral blood samples, and B- and T-cell concentraons in human peripheral blood. Three cell counng methods are compared: the hema- cytometer, counng bead frequency in flow cytometric samples, and direct sample volume measurement by a flow cytometer. No significant difference was found in the average cell count per µL of sample deter- mined by the three counng methods. However, significant differences were found when the precision of the cell count data for the hemacy- tometer and counng bead methods was compared to direct volume measurement by a flow cytometer. Not surprisingly, hemacytometer counts, including trypan blue for viability assessment, had the largest variability between quadruplicate counts on the same sample (average CV = 19.5%). The combined use of counng beads and the flow cytom- eter improved counng precision a great deal (average CV = 2.94%) over the hemacytometer method. However, the most precise measurement was obtained by direct volume measurement with the flow cytometer (average CV = 2.1%), with p values of 0.002 and 0.010 when compared to the hemacytometer and counng bead methods, respecvely. This study shows that a benchtop flow cytometer with tradional laminar flow fluidics and direct volume measurement capabilies, is equally ac- curate and more precise, than either hemacytometer or counng bead methods for determining cell concentraon for applicaons as diverse as cultured cell line viability or human platelet counts. The development of a flow cytometer with a unique peri- stalc pump fluidics system allows direct measurement of the volume sampled from cell suspensions. The system operates with standard laminar flow crucial to accurate fluorescence and light scaer measurements, and has no sample volume limit. Cell concentraon measurements determined by the direct volume method gave comparable results to those obtained with standard methods (hemacytometer and counng beads). The direct volume measurement method was significantly more precise for triplicate counts of the same cell sample than either the hemacytometer (p=0.002) or bead count (p=0.010) methods. Hemacytometer counts can be highly imprecise (average CV for triplicate counts = 19.51%). In contrast, flow cytometers can funcon as precision cell counng tools and offer the addional advantage of mul- parametric sample analysis. Flow cytometers offer a fast, precise alternave to hemacy- tometers for viable cell counng. Direct idenficaon and counng of T- and B- cell popula- ons is quickly and easily achieved by flow cytometry. Accurate platelet counng is enabled through fluorescence signal triggering on a flow cytometer. Small parcles, such as platelets cannot be effecvely counted using a hemacy- tometer. The addion of counng beads to every flow cytometric sample saved me and improved counng precision com- pared to hemacytometer counts, but added expense to the analysis. The direct-volume measurement method was the most pre- cise and least me-consuming approach for cell counng. Clare Rogers, Maria Dinkelmann, Nathan Bair, Collin Rich, Grant Howes, Brett Eckert · Accuri Cytometers, Inc. Ann Arbor, MI Comparison of Three Methods for the Assessment of Cell Phenotype, Viability, and Concentration in Cultures and Peripheral Blood Historically, the combinaon of a light microscope and hemacytometer have been the tools of choice for performing cell counts in life science research laboratories. However, this method is slow and prone to error. When hemacytometer counts and flow cytometric (FC) phenotypic data are combined to determine cell subset numbers, errors are mulplied. Digital flow cytometers, ulizing laminar-flow fluidics, allow fast, phenotypic data collecon (at rates up to 10,000 events per second) on a wide range of cell types (sub-micron sized bacteria through large mammalian cell lines) but sll require the addion of counng beads to each sample to calculate cell-subset concentraon. On the other hand, flow cytometers with syringe driven fluidics can deliver absolute count measurements without the addion of counng beads to samples, but are oſten limited by lower data acquision rates (< 1,000 events per second) and the total volume of sample that can be analyzed from each sample tube. We have developed a fully-digital flow cytometer with a unique peristalc pump driven, laminar-flow fluidics system (Figure 1). This combines the advantages of hydrodynamically focused cell sampling (high data acquision rates and good light scaer and fluorescence resoluon) with the ability to calculate absolute counts for any idenfied populaon in a sample. Several applicaons of direct cell concentraon determinaon based on volume measurement are presented here: viability assessment of cultured cell lines, platelet counts for whole, human peripheral blood samples, and determinaon of T- and B-cell numbers in human peripheral blood. All methods make use of the fluorescence and light scaer measurements possible with a flow cytometer to idenfy sub-populaons of interest. Cell counng data collected by at least one other tradional method (counng beads or hemacytometer) is included for comparison. Introduction Abstract Cell Counting Method 1 2 3 Coefficient of Variation 0 10 20 30 40 direct-volume (C6 cytometer) counting beads (C6 cytometer) Hemacytometer p=0.002 p=0.010 Figure 1 Figure 2 Figure 3 Cell viability was measured in high and low density cultures of three cell lines: U937 (human lymphoma), Jurkat (human T-cell leukemia) and NIH/3T3 (mouse fibroblast) using three different methods. 7-AAD (Cayman Chemicals) and trypan blue (HyClone) exclusion were used to determine viability according to manufacturers’ instrucons. Figure 4 Figure 5 B. A. C. A. B. C. D. 0 500000 1000000 1500000 2000000 2500000 Viable Cell Number volume counting beads hemacytometer U937 saturated U937 low density Jurkat saturated Jurkat low density 3T3 low density 3T3 saturated D . . . . . . . . . . . Figure 6 An example of viable cell gate (P5) placement using un- stained control cells. An example of dead cell gate (P6) placement using 7-AAD posive cells. 1.0 µL 7-AAD was added to 500 µL cell sus- pension and incubated in the dark at room temperature for 15 minutes. 7-AAD is a fluorescent dye which is exclud- ed from viable cells, but taken up by cells when the outer membrane is compromised (as in cell death). Viable and dead cell concentraons for high density cell samples of three cell types as measured by gated cell counts relave to volume sampled. Comparison of mean viable cell counts (and standard deviaon) for three methods of cell counng using high and low density cultures of three different cell lines. Beads 2009 © Concentraons of B- and T-cell sub-populaons in human peripheral blood are measured using a flow cytometer. 50 µL of human peripheral blood from three donors was labeled with 5 µL each of FITC-CD3 (eBioscience) and PE-CD19 (Biolegend) for 20 minutes at room temperature in the dark. Subsequently, red blood cells were lysed with BD FACS Lyse according to manufacturer’s in- strucons. Cells were pelleted, and resuspended in 500 µL PBS + 5% BSA, and 50 µL of SPHERO TM AccuCount counng beads were added to each tube. Triplicate readings were taken of each sample. B- and T-cell populaons were idenfied by CD19 and CD3 posive cells respecvely. B- and T-cell concentraons were calculated using volume and count bead methods. B. A. Counting Beads CD3-FITC FL1 CD19-PE FL2 13.6% 58.8% average T-cell counts/ul (%CV) average B-cell counts/ul (%CV) vol method bead method vol method bead method donor 1 113 (0.9%) 112 (2.1%) 24 (4.8%) 24 (5.3%) donor 2 297 (1.7%) 281 (3.3%) 69 (1.5%) 65 (3.2%) donor 3 126 (2.1%) 121 (2.2%) 19 (7.2%) 18 (4.7%) Light Scaer Gate = Lymphocytes + Beads B A y = 1.116x r² = 0.9774 n = 75 1,000 10,000 100,000 1,000,000 10,000,000 1,000 10,000 100,000 1,000,000 10,000,000 Counting Bead: cell count per mL Direct Volume: cell count per mL sample
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Validation of direct volume cell concentration measure-ments using counting beads.
Viable cell concentration measurements are similar using direct volume, counting bead or hemacytometer methods.
Platelet concentrations in human blood samples are obtained using fluorescence triggering on a flow cytometer.
The direct volume method is more precise for measuring cell concentration than counting bead or hemacytometer methods.
Peristaltic pumps allow direct cell concentration measurements and high data acquisition rates.
Development of a unique peristaltic pump-driven laminar flow fluidics system combines the advantages of hydro-dynamically focused cell sampling (high acquisition rates, good light scatter and fluorescence resolution) with the ability to report absolute cell counts for any identified population in a sample.
Comparison of cell concentration measurements by two methods. The x axis represents cell concentration calculated by absolute cell counts in relation to volume sampled. The y axis represents cell concentration as calculated by the absolute cell counts in re-lation to the number of SPHEROTM AccuCount beads measured. 75 different samples were analyzed in five independently run experi-ments. Cell types analyzed included human peripheral blood sam-ples and mammalian cell lines.
The average coefficient of variation for replicate cell counts using three different counting methods on the same samples. A paired student’s T test was used to determine p values (95% confidence, N = 23). The direct-volume measurement method provides the least variability between replicate cell counts (average CV values: direct-volume measurement =2.10%; counting bead method =2.94%; hemacytometer =19.51%).
Aliquots of whole blood were diluted 1:10 into HEPES-buffered saline with 1% formaldehyde. 20 µL aliquots of diluted blood were incubated with 20 µl CD41-PE antibody (DAKO clone 5B12) for 20 minutes in the dark at room temperature. Samples were then diluted with 1 mL HEPES-buffered saline with 1% formaldehyde. 5.0 µL of RFP-50-5 beads (Spherotech) were added to allow comparison of two counting methods.
Triggering data collection on forward scatter.
Same sample triggered on fluorescence, showing improved discrimination of platelets.
Comparison of platelet counts obtained for two methods of counting. All data was collected using CD41-PE FL2 Trigger.
Program #2785
Results
Conclusions
Technology giving researchers the ability to quickly and precisely deter-mine the absolute number or concentration of cells with a given pheno-type is of great interest in diverse fields including clinical research and diagnostics, drug discovery, and cell biology. The recent advent of small, fully digital flow cytometers combines the advantages of hydrodynami-cally focused cell sampling, high data acquisition rates and excellent light scatter resolution with the ability to calculate absolute counts for any identified population in a sample. In the study presented here, sev-eral different cell counting applications are explored: viability determi-nation for cultured cell lines, platelet counts in whole, unlysed human peripheral blood samples, and B- and T-cell concentrations in human peripheral blood. Three cell counting methods are compared: the hema-cytometer, counting bead frequency in flow cytometric samples, and direct sample volume measurement by a flow cytometer. No significant difference was found in the average cell count per µL of sample deter-mined by the three counting methods. However, significant differences were found when the precision of the cell count data for the hemacy-tometer and counting bead methods was compared to direct volume measurement by a flow cytometer. Not surprisingly, hemacytometer counts, including trypan blue for viability assessment, had the largest variability between quadruplicate counts on the same sample (average CV = 19.5%). The combined use of counting beads and the flow cytom-eter improved counting precision a great deal (average CV = 2.94%) over the hemacytometer method. However, the most precise measurement was obtained by direct volume measurement with the flow cytometer (average CV = 2.1%), with p values of 0.002 and 0.010 when compared to the hemacytometer and counting bead methods, respectively. This study shows that a benchtop flow cytometer with traditional laminar flow fluidics and direct volume measurement capabilities, is equally ac-curate and more precise, than either hemacytometer or counting bead methods for determining cell concentration for applications as diverse as cultured cell line viability or human platelet counts.
The development of a flow cytometer with a unique peri-staltic pump fluidics system allows direct measurement of the volume sampled from cell suspensions.
The system operates with standard laminar flow crucial to accurate fluorescence and light scatter measurements, and has no sample volume limit.
Cell concentration measurements determined by the direct volume method gave comparable results to those obtained with standard methods (hemacytometer and counting beads).
The direct volume measurement method was significantly more precise for triplicate counts of the same cell sample than either the hemacytometer (p=0.002) or bead count (p=0.010) methods.
Hemacytometer counts can be highly imprecise (average CV for triplicate counts = 19.51%).
In contrast, flow cytometers can function as precision cell counting tools and offer the additional advantage of multi-parametric sample analysis.
Flow cytometers offer a fast, precise alternative to hemacy-tometers for viable cell counting.
Direct identification and counting of T- and B- cell popula-tions is quickly and easily achieved by flow cytometry.
Accurate platelet counting is enabled through fluorescence signal triggering on a flow cytometer. Small particles, such as platelets cannot be effectively counted using a hemacy-tometer.
The addition of counting beads to every flow cytometric sample saved time and improved counting precision com-pared to hemacytometer counts, but added expense to the analysis.
The direct-volume measurement method was the most pre-cise and least time-consuming approach for cell counting.
Clare Rogers, Maria Dinkelmann, Nathan Bair, Collin Rich, Grant Howes, Brett Eckert · Accuri Cytometers, Inc. Ann Arbor, MI
Comparison of Three Methods for the Assessment of Cell Phenotype, Viability, and Concentration in Cultures and Peripheral Blood
Historically, the combination of a light microscope and hemacytometer have been the tools of choice for performing cell counts in life science research laboratories. However, this method is slow and prone to error. When hemacytometer counts and flow cytometric (FC) phenotypic data are combined to determine cell subset numbers, errors are multiplied. Digital flow cytometers, utilizing laminar-flow fluidics, allow fast, phenotypic data collection (at rates up to 10,000 events per second) on a wide range of cell types (sub-micron sized bacteria through large mammalian cell lines) but still require the addition of counting beads to each sample to calculate cell-subset concentration. On the other hand, flow cytometers with syringe driven fluidics can deliver absolute count measurements without the addition of counting beads to samples, but are often limited by lower data acquisition rates (< 1,000 events per second) and the total volume of sample that can be analyzed from each sample tube.
We have developed a fully-digital flow cytometer with a unique peristaltic pump driven, laminar-flow fluidics system (Figure 1). This combines the advantages of hydrodynamically focused cell sampling (high data acquisition rates and good light scatter and fluorescence resolution) with the ability to calculate absolute counts for any identified population in a sample.
Several applications of direct cell concentration determination based on volume measurement are presented here: viability assessment of cultured cell lines, platelet counts for whole, human peripheral blood samples, and determination of T- and B-cell numbers in human peripheral blood. All methods make use of the fluorescence and light scatter measurements possible with a flow cytometer to identify sub-populations of interest. Cell counting data collected by at least one other traditional method (counting beads or hemacytometer) is included for comparison.
Introduction
Abstract
Cell Counting Method1 2 3
Coe
ffici
ent o
f Var
iatio
n
0
10
20
30
40
direct-volume(C6 cytometer)
counting beads(C6 cytometer)
Hemacytometerp=0.002
p=0.010
Figure 1 Figure 2 Figure 3
Cell viability was measured in high and low density cultures of three cell lines: U937 (human lymphoma), Jurkat (human T-cell leukemia) and NIH/3T3 (mouse fibroblast) using three different methods. 7-AAD (Cayman Chemicals) and trypan blue (HyClone) exclusion were used to determine viability according to manufacturers’ instructions.
Figure 4 Figure 5
B.A.
C.
A.
B.
C.
D.
0
500000
1000000
1500000
2000000
2500000
Viab
le C
ell N
umbe
r
Comparison of Viable Cell Counting Methods
volume
counting beads
hemacytometer
0
500000
1000000
1500000
2000000
2500000
Viab
le C
ell N
umbe
r
Comparison of Viable Cell Counting Methods
volume
counting beads
hemacytometer
U937
satu
rated
U937
low d
ensit
y
Jurk
at sa
tura
ted
Jurk
at lo
w den
sity
3T3 low
den
sity
3T3 sa
tura
ted
D
.
.
.
.
.
.
.
.
.
.
.
Figure 6
An example of viable cell gate (P5) placement using un-stained control cells.
An example of dead cell gate (P6) placement using 7-AAD positive cells. 1.0 µL 7-AAD was added to 500 µL cell sus-pension and incubated in the dark at room temperature for 15 minutes. 7-AAD is a fluorescent dye which is exclud-ed from viable cells, but taken up by cells when the outer membrane is compromised (as in cell death).
Viable and dead cell concentrations for high density cell samples of three cell types as measured by gated cell counts relative to volume sampled.
Comparison of mean viable cell counts (and standard deviation) for three methods of cell counting using high and low density cultures of three different cell lines.
Beads
2009©
Concentrations of B- and T-cell sub-populations in human peripheral blood are measured using a flow cytometer.
50 µL of human peripheral blood from three donors was labeled with 5 µL each of FITC-CD3 (eBioscience) and PE-CD19 (Biolegend) for 20 minutes at room temperature in the dark. Subsequently, red blood cells were lysed with BD FACS Lyse according to manufacturer’s in-structions. Cells were pelleted, and resuspended in 500 µL PBS + 5% BSA, and 50 µL of SPHEROTM AccuCount counting beads were added to each tube. Triplicate readings were taken of each sample.
B- and T-cell populations were identified by CD19 and CD3 positive cells respectively.
B- and T-cell concentrations were calculated using volume and count bead methods.
B.
A.
Counting Beads
CD3-FITC FL1
CD
19
-PE F
L2 13.6%
58.8%
average T-cell counts/ul (%CV) average B-cell counts/ul (%CV)vol method bead method vol method bead method
donor 1 113 (0.9%) 112 (2.1%) 24 (4.8%) 24 (5.3%)donor 2 297 (1.7%) 281 (3.3%) 69 (1.5%) 65 (3.2%)donor 3 126 (2.1%) 121 (2.2%) 19 (7.2%) 18 (4.7%)
Light Scatter Gate = Lymphocytes + Beads
BA
y = 1.116xr² = 0.9774
n = 75
1,000
10,000
100,000
1,000,000
10,000,000
1,000 10,000 100,000 1,000,000 10,000,000
Cou
ntin
g B
ead:
cel
l cou
nt p
er m
L
Direct Volume: cell count per mL sample
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