Quantitative assessment of cell viability based on flow cytometry and microscopy.
We compare flow cytometric and microscopic determination of cell viability by fluorescence labeling using calcein acetoxy-methyl-ester and ethidium homodimer-1 as live and dead stain, respectively. Peripheral blood monocytes served as model system and were accumulated applying density gradients. Subsequently, monocytes were further enriched by magnetic-activated or fluorescence-activated cell sorting (MACS, FACS) targeting the antigen CD14. Identical samples were used for flow cytometric and microscopic analysis to allow direct comparison of both analysis methods. More than 1,000 cells were measured for each sample to minimize the measurement uncertainty caused by counting statistics. We observed good agreement of flow cytometric and microscopic viability measurements. On average, the difference in viability measured by flow cytometry and microscopy amounted to (2.7 ± 1.4)% for live staining and (1.7 ± 1.2)% for dead staining. These deviations were similar to the uncertainty of measurement for cell viability, thus demonstrating that both methods delivered equal results. Besides monocytes, comparison of flow cytometric and microscopy viability for MACS enriched CD34-positive cells also showed consistent results.
Physikalisch-Technische Bundesanstalt, 10587 Berlin, Germany. andreas.kummrow@ptb.de
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A
Kummrow
M
M
Frankowski
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N
Bock
C
C
Werner
T
T
Dziekan
J
J
Neukammer
D000328:Adult; D002469:Cell Separation; D002470:Cell Survival; D004996:Ethidium / Q000031:analogs & derivatives / Q000737:chemistry; D005260:Female; D005434:Flow Cytometry; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008297:Male; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D009000:Monocytes / Q000502:physiology*; D013194:Staining and Labeling
D005452:Fluoresceins; D005456:Fluorescent Dyes; D018950:Lipopolysaccharide Receptors; C085925:calcein AM; C018533:ethidium homodimer; D004996:Ethidium