Development of a Direct Trypan Blue Exclusion Method to Detect Cell Viability of Adherent Cells into ELISA Plates

Abstract

Cell viability detection is important in cell culture applications including measurement of cell proliferation i.e for understanding cytotoxic effects of compounds on cells. There are some cell viability methods based on fluorescence or non-fluorescence detection. More simplified evaluation for cell viability, such as trypan blue staining, can be preferred before performing fluorescence assays. This appears advantageous when to have a large number of cell samples in ELISA plates after treatments with different concentrations of drug candidates. Thus, further fluorescence assays can include less concentrations rather than experiencing all used along 96-well plates. For this, trypan blue exclusion method is an option. Traditionally, treated cells are harvested by centrifugation and incubated with trypan blue within tubes followed by transferring the mixture into a hemacytometer with two chambers and assessed under the microscope. Nevertheless, using a hemacytometer limits practicability of this method when analyzing various cell samples into 96-well plates at the same time. This study was aimed to adapt trypan blue method to in situ staining of adherent cells cultured on ELISA plates. For this, cells were fixed with different fixatives after trypan blue incubation to maintain cells in impenetrable meshwork, and paraformaldehyde was the most effective fixative. This modified protocol was validated by testing the effect of dimethylsulfoxide-a cytotoxic agent-on cells, and expectedly found that cell viability reduced with higher concentrations of dimethylsulfoxide suggesting that in situ detection of cell viability by trypan blue can be a useful tool for preliminary detection of cells cultured on ELISA plates before performing automatized experiments with such flow cytometer and/or microplate reader.

Keywords

• Cell viability,cell death,adherent cells,trypan blue exclusion,ELISA plate,fixation

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