Quantitative assessment of cell viability based on flow cytometry and microscopy

Abstract

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 AE 1.4)% for live staining and (1.7 AE 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. ' 2012 International Society for Advancement of Cytometry

A Biospecimen Proficiency Testing Program for Biobank Accreditation: Four Years of Experience

“…Flow cytometry requires highly trained operators and expensive systems. 23 Small variations have been observed, as trypan blue dye generally provides higher viability proportion than flow cytometry. 24 These discrepancies may be explained by the individual uncertainties of microscopic examination and the lower sensitivity of optical microscopy to detect unstained cells.…”

“…Both factors may then lead to overestimation of viable cells by trypan blue determination. 23 However, when a sufficiently high number of cells were counted, good agreement has been shown between trypan blue exclusion and flow cytometry measurements. 23,25 This was confirmed by the results of the PT program, as no significant difference was observed between trypan blue and flow cytometry results.…”

“…Cellometer (Nexcelom Bioscience) is based on the same principle as trypan blue, but is automated and more precise. 23 Flow cytometry gives a highspeed analysis of cells in suspension, with a distinction between viable and nonviable cells. Flow cytometry requires highly trained operators and expensive systems.…”

Automated cell viability assessment using a microfluidics based portable imaging flow analyzer

“…1 Both flow cytometry and microscopy are gold-standard techniques employed in medical diagnostics as well as basic biological research. Flow cytometry enables automated high-speed quantitative multiparameter analysis of large population of cells.…”

“…[1][2][3] Viability testing is performed to assess the live/dead condition of cells present in a given population and infer cell survival rate as a result of cytotoxicity, measure cell morbidity in apoptosis, potential of bacteria to survive an environmental shock, etc. Morphological changes or changes in membrane permeability are used to asses the viability of cells.…”