Hi all,
I have been working on WST-1 cytotoxicity assay for my anti-cancer drug screening project. However, there is an issue that the OD value obtained is not consistent with what I can see under the microscope. This issue is apparently associated with both cell line and drug:
-With the same drug tested, two cell lines giving similar observation under the microscope give very different relative activity from the WST-1 assay. One gives consistent low OD value after drug treatment and one gives OD value similar to the control, which is growing happily and without cell death. This problem of inconsistency is particularly common in hepatocarcinoma cell lines.
-With the same problematic cell line mentioned above, the result of cisplatin (which is included as a control of toxic compound) is completely fine. Cell death is accompanied with low OD value. However, the compounds that I am testing give similar response in cell death but with an OD value very close to the vehicle control.
The experimental set-up is as follow:
Day 1: 6000 cells in 50ul medium per well are seeded into the 96-well plates
Day 2: drugs are diluted in 50ul medium per well and added to the cells
Day 4: medium with drugs are removed. WST-1 and PMS are diluted in 10ml of medium without serum. 100ul of the mixture is added into each well.
Is there any suggestions to solve the problem? Thanks a lot!
-MC
Dear Christina,
Thanks for your reply!
I also suspect that was due to the residual enzymatic activity remaining in the dead cells. However, cisplatin also causes severe cell death in the same cell lines and the resulted OD values are indeed close to the background. This confuses me a lot.
Moreover, at the cytostatic concentrations of the candidate compounds, the cell number is around 1/3 to 1/4 of the control without severe cell death. The OD value in these wellls are also close to the control which I have no idea how to explain.
-MC
Dear Christina,
I usually start measuring the OD at 1 hour after addition of WST-1 to the cells. If the OD value is too low, I would take another measurement at around 1.5 to 2 hours. The OD values that I obtained in the aforementioned experiments were only around 0.4-0.7 for different cell lines, which they can give a maximum OD of more than 1.2 if I allow a longer incubation. From this, I would guess the cells haven't started to die due to the effect of WST-1.
Anyway, I would try measuring starting from 30min after addition next time to see if that would make a difference.
Thank you!
-MC