I am trying to undergo a cell viability assay with trypan blue staining. I originally got cell suspension by pipetting and observe cells after staining on the hemocytometer.
But I found out my cell seems too fragile after treatment, therefore, I can't see any of the cells under the microscope. (but the cells in the control group are still visible under the microscope, and the cells in the treatment group are still visible in the original wells.)
Thus, I would like to ask if anyone tried to stain cells with trypan blue directly in the well?
Thanks a lot for you answering it.
Hello Lisa Liu
Staining of cells with trypan blue directly in the wells would not be proper.
Usually, in trypan blue exclusion assay of cell viability, the cell suspension is diluted with trypan blue 0.4% solution in the ratio 1:1, and cells are counted using hemocytometer. The hemocytometer will give you the accurate cell count using the formula below.
The cell count per ml is determined by
Average cell count from the four corner squares x dilution factor x 10^4 cells /ml
When you perform cell viability assay using trypan blue, you determine percent viability of cells that are viable and in healthy condition. In your case, during treatment, the cells become fragile which means that they are not in healthy condition, and when you pipette, these cells are stressed and eventually they may be dying. Even though the cells in the treatment group are still visible in the original wells, they are not in good condition and cannot be considered as viable. So, staining cells with trypan blue directly in the well will give you results that can be misleading.
Best.
Hi, Lisa Liu !
While staining cells directly in the wells is not recommended; you may attempt it by carrying out the identical procedure with your control group of cells as well. However, if I may suggest; try limiting your trypan blue treatment to 3.5 to 5 minutes only before attempting to count in the well itself? Sometimes cells which are too fragile still stay intact on reducing the treatment time with trypan blue (you need a minimum of 3.5 minutes for cells to permeate membranes at all) to ~5min instead of 10.
If you do attempt to count in the well itself, do count at least ~100 cells/ well (without double counting the cells) for your count to hold any value. While a minimum of triplicate in experiments is always necessary, since you mentioned the fragility of cells, do increase the number of repeats for this test to minimise bias which can then affect your future work.
An alternative to this might be utilising alternative viability tests such as resazurin/crystal violet/ MTT etc, which are colorimentric in nature and eliminate the possibility of cell loss since the staining occurs in the same well as the treatment.
Hope this helps, all the very best!
If you are comparing control and treated adherent cells, and your cultures are not too dense to count, staining and counting in the dish is fine:
Article In Situ Trypan Blue Staining of Monolayer Cell Cultures for ...