Comparison of the automated fluorescence microscopic viability test with the conventional and flow cytometry methods

Abstract

The cell viability test is an essential tool in any laboratory, performing cell-based studies and clinical laboratory tests. The trypan blue exclusion method is the most popular assay for its simple concept among various diagnostic tools. However, several disadvantages include time-consuming and labor-intensive steps with low precision. In this study, we evaluated a new technique for the automatic cell viability measurement with microscopic cell counter and microchip. Upon blood draw from 11 healthy volunteers, Mononuclear cells were separated immediately from the heparinized whole blood, and the viable cells were diluted from 100 to 1%. The cell viability tests were performed simultaneously with following three methods: the conventional manual trypan blue exclusion method; the flow cytometry measurement with propidium iodide stain; and the newly developed microscopic cell counter with microchip. Linearities, precisions, and correlations from three methods were analyzed and compared. The correlations data from the microscopic cell counter were in good agreement with both the conventional trypan blue method (r=0.99, P<0.05) and the flow cytometry (r=0.99, P<0.05), respectively. The precision (2.0-6.2%) and linearity from the microscopic cell counter method with microchip were superior in comparison with the conventional method. The microscopic cell counter with microchip performed well with high precision, linearity, and efficient running time than both the manual trypan blue and the flow cytometry methods.

Trypan blue exclusion assay by flow cytometry

“…A previous study (9) showed that live cells in contact with 0.4% TB for more than 5 min may incorporate the dye, leading to overestimation of the number of dead cells. We therefore evaluated whether the optimum TB concentration for use with flow cytometry had a similar imprecision.…”

A luciferase-based assay for rapid assessment of drug activity against Mycobacterium tuberculosis including monitoring of macrophage viability

“…Intrinsic methods e.g. the GFP microplate assay (Collins LA and Franzblau, 1998) and auto-luminescent mycobacteria expressing the lux operon (Andreu et al, 2010) enable repeated measurements in contrast to endpoint assays like CFU plating and other methods as the Microplate Alamar Blue Assay (Kim et al, 2011). Another advantage of the luciferasebased method is its higher signal to noise ratio as described (Collins, 1997).…”

A new system for quality control in hematopoietic progenitor cell units before reinfusion in autologous transplant

“…The NucleoCounter NC‐3000 enables automated cell counting and analyses of a wide number of samples to be performed at the same time; it is easy to use and also guarantees an excellent precision reducing the data variability due to the operator's work. Other tests used in the clinical routine, such as the determination of cell viability with trypan blue, are characterized by a huge variability of the results 28‐30…”