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

Year 2018, Volume: 14 Issue: 1, 99 - 104, 30.03.2018

Selcen Çelik Uzuner

https://doi.org/10.18466/cbayarfbe.372192

Cited By: 11

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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