ANALYTICAL AND LABORATORY EQUIPMENT 17
Improved cell counting technology F
Clare Rogers and MaryAnn Labant look at the use of flow cytometers for cell counting.
or years, cell counts in life science research laboratories have been performed
using a light microscope and hemacytometer. Tis method is slow and prone to error. Errors multiply when hemacytometer counts and flow-cytometric (FC) phenotypic percentages are combined to determine cell subset numbers. Digital flow cytometers, utilising laminar-flow fluidics, allow fast, multiparametric data collection (up to 10 000 events per second) on a wide range of cell types but require addition of counting beads to each sample to calculate cell-subset concentration.
Alternatively, 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 (<1000 events per second), diminished fluorescence and
Software Statistics Table reports events/ μL for viable (P5) and dead (P6) cells for each triplicate sample containing
7-AAD. (D) Comparison of mean viable cell count (± CV) for three methods of counting.
sample. (B) Setting dead-cell gate (P6) using positive 7-AAD sample. (C) CFlow
Fig. 1. Viable cell count determinations with the C6: (A) Setting viable-cell gate (P5) using negative 7-AAD control
light-scatter resolution, and a propensity for flow-cell clogging. Flow cytometers with unique peristaltic pump-driven, laminar- flow fluidics systems combine the advantages of hydrodynamically focused cell sampling (high data- acquisition rates, good light scatter and fluorescence resolution) with the ability to report absolute counts for any identified population in a sample.
Two applications of direct cell- concentration determination are presented that make use of the fluorescence and light-scatter measurements possible with a flow cytometer to identify sub- populations of interest. Counting bead or hemacytometer data is included for comparison.
Viability assessment Initial cultures of U937 and Jurkat cell lines were seeded so that the final analysis cell concentration was
dense or light. After one week of culture, cells were resuspended and 1mL transferred into a 1.5mL tube. 7-Amino-actinomycin D (7-AAD, Cayman Chemical), a fluorescent dye which is excluded by viable cells, was used as a marker of cells with compromised outer membranes.
5.0µL of a 1mg/mL 7-AAD solution, along with 50µL of AccuCount Fluorescent Particles (Spherotech; ACFP-50-5) were added to each sample tube and mixed thoroughly. Tubes were kept at room temperature (RT) in the dark, and sampled between five and 30 minutes after addition of 7-AAD, with gentle mixing immediately prior to analysis.
Microscopic cell counts were performed with a hemacytometer. Appropriate dilutions of samples were made into phosphate-buffered saline containing Trypan Blue. Triplicate counts of at least 100 non-
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