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 96well 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.

Effect of Rehmannia glutinosa Libosch extract on proliferation and cardiogenic pre-differentiation of human mesenchymal stem cells

“…The viability rate of UC-MSCs was evaluated through Trypan blue assay like afore-research [ 20 ]. After the above incubation, the culture medium was removed, and these cells were exposed to serial 2-fold dilution of RGE (50 ppm, 100 ppm, 200 ppm, 400 ppm, 800 ppm, 1600 ppm, 3200 ppm, 6400 ppm) for 24 h. After treatment, the treated and untreated cells were stained with a previous procedure [ 21 ]. Briefly, these cells were incubated with 0.4% trypan blue for 10 min at room temperature (RT) after three washing times.…”

Enhanced Antiobesity Efficacy of Tryptophan Using the Nanoformulation of Dendropanax morbifera Extract Mediated with ZnO Nanoparticle

“…To detect cell viability, direct trypan blue exclusion method was used as proposed by Selcen et al, 2017 [ 24 ]. Cells were treated in a 6-well plate with DM-ZnO-Try NE samples for 3 h and 24 h at the concentrations 10 μg/mL and 12 μg/mL.…”

Immune Suppression Induced by Gallibacterium anatis GtxA During Interaction with Chicken Macrophage-Like HD11 Cells

“…Trypan blue viability measurement was performed by standard method [14,44,45]. The 24-well plates, containing 0.7 × 10 6 cell/well, were infected with G. anatis 12656-12 as invasion assay described above.…”