I followed the protocol to dilute 0.4% Trypan Blue solution 1:1 with cell suspension (dilute in 0.9% NaCl). And did the observation with a light microscope, no dead cells/blue cells were detected also after exceeding 30 minutes of incubation. Cells stay unstained although different methods were used to create dead cells.
Thanks for the answer,
but exceeding 30 mins incubation time was only one procedure to decrease the cell viability following the protocol "http://www.lonza.com/products-services/bio-research/cell-culture-products/reagents/cell-culture-reagents/trypan-blue-04-solution.aspx"
I used Trypan Blue to stain several whole blood dilutions/cell suspensions that is assumed to have at least some dead RBCs. But all of them stay unstained, so i want to make sure that i did a right differentiation and create whole blood dilutions that underwent treatments that damage the cells, but no dead cells/ blue stained cells could be observed. So I am not sure what i did wrong during the staining procedure.
It is somehow weird that it does not stain the dead cells because the trypan blue should be able to pass through the cell membrane when the cell is dead.
Maybe as a positive control you could really kill all of your cells by adding staurosporine or sonication. Then you should definitely see dead cells.
Or you could try another assay to evaluate cell viability, such as Prestoblue or MTT.
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