i want to determine the cytotoxicity activities of some drugs with WST-1 assay using 293T cell lines in 96 well plate. I usually seed 10,000 cell per well and incubate for ~48 hours.
I expected the cell viability to decrease with increase in drug doses, however, I discovered that the readings are not consistent at all. for instance I can't explain why 10microMol of Hydrogen peroxide will be more toxic than 50microM and same observation with other drugs. I also expect the negative control to have the highest reading (@450nm) but that has not been the case.
I also discovered this morning that the 293 cells are easily washed out when I apply PBS prior to adding new medium + wst-1 reagent. Since I am testing the cytotoxicity against time, I have to wash the drugs after stipulated times (e.g. 1h, 2h, 4h, 6h, and 12h) and introduce new medium + wst-1 reagent.
I don't really know what to do next as I have troubleshoot severally.
Please, any suggestion ?
Thanks
Hello Olatunde Omotoso
In WST-1 assay, WST-1 is converted to the corresponding formazan by cellular mitochondrial dehydrogenases. The more the number of viable cells, the higher will be the dehydrogenases activity and so the amount of formazan product formed will be high. However, the presence of residual enzyme activity in the dead cells present in the wells could also contribute to the final absorbance value. This probably could be causing the inconsistency that you observe in WST-1 assay .
Further, I would suggest you reduce the seeding density per well. You could seed 5000-6000 cells per well.
Regards.
Hi Olatunde Omotoso
Try those tips:
1) Don't seed the external wells as they are more subjected to the heat of the incubator, thus more evaporation of media, and eventually variation in your readout. Put on those external wells water or PBS or media.
2) for the same reason of heat effect, I prefer seeding the cells in 150 or 200ul rather than 100ul.
3) When you remove the media be very gentle, and try to withdraw the maximum amount from the edge of the well, rather than the center. For example, if you have the cells in 200ul, try removing only 180 ul to avoid disrupting the cell monolayer.
4) When using 96-well plate or small volumes in general, try using the reverse pipetting technique to maintain very low variation between wells.
Hope this would help.
Alaa Refaat , Thank you for the suggestion, I have actually noticed and wondered that the volume of the media usually reduce overtime but I never thought that evaporation could be the cause. I will try this out
Malcolm Nobre , don't you think seeding 5000 per well would be too little for a cytotoxic assay that is expected to cause cell mortality? I would appreciate your further explanation on this
Thank you very much
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