Do you count the cells after each step of the protocol as these type of cells are floating cells and I have a feeling that I am losing a big number while washing, so I end up with various of different absorbances in every triplicate wells.
Hi Dima,
I suggest you not to wash your cells and be gentle as possible as you can.
Count your cells at the beginning of the experiment and inoculate them one day before the treatment or directly inoculate them with your treatment whatever it is (drug etc). If you prefer the incubate your cells one day, so use only 100 ul medium for culture. And the treatment day, add another 100 ul medium with your treatment very gently without disturbing the cells (dropwise).
Incubate the cells with treatment for a period of time. For the fixation of the cells, use cold 80-100%TCA as a stock solution to reach final concentration 16-20% TCA by directly adding the cold 80-100% TCA into the wells (e.g. If your well contains 200 ul medium, gently add 50 ul 80-100%TCA to reach 16-20% TCA final without disturbing your cells, if you do not add TCA gently, cells will be dislodged so your results will be affected so you can't read same OD from same conditions). After fixation, cells must be fixed on the surface of the plate and you can proceed the protocol as the usual wash and staining steps.
Good luck
Thank you very much, the thing is that I am centrifuging with 1800 rpm after each step, and yes I am adding 100 ul of 20% TCA to 200 ul the final volume in each well on plate, I believe that centrifuging might be disturbing my cells?
You are welcome, and I believe centrifugation could affect the cells. Try do everything without centrifuge steps.
Best
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