I have seeded HeLa cells in a calculation of 10000 cells per well in a 96 well plate. Then I went for treatment in the next day. Then at last, I added 7ul of 5mg/ml solution of MTT reagent into each well (Point to note: without removing the previous media, the MTT was added) but the absorbance values post addition of DMSO was really less and not significant.
My seniors from different lab told me that:
1. In the treatment day, when we have to add media+drug into each well after removing the previous day media, I am removing the cells as well
2. In the last day, post MTT addition when formazan crystals are formed, dissolved in DMSO solution, I am removing the crystals as well.
Please help me out with this issue. I have been repeating MTT expt for around 8th time. Thanks and regards.
ad 1): What does a microscopic view of the cultures tell you? Have you counted the cells? Are they well attached?
ad 2): Besides checking your cell numbers, the MTT assay reflects metabolic activity, and may provide varying results depending on the compounds added to the cells.
see:
- Vistica, D.T.; Skehan, P.; Scudiero, D.; Monks, A.; Pittman, A.; Boyd, M.R. Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production. Cancer Research 1991, 51, 2515-2520.
- Ulukaya, E.; Colakogullari, M.; Wood, E.J. Interference by anti-cancer chemotherapeutic agents in the MTT-tumor chemosensitivity assay. Chemotherapy 2004, 50, 43-50.
Hope this well help you.
a1) The microscopic view was well and good, not that everything is just vanished. Yeah they showed their proper morphology post attachment.
a2) Yeah correct, I was skeptical with the addition of MTX, atleast that should have given me the std. results.
Sure thanks for sharing, I'll go through.
Most solvents used to dissolve the crystals do not completely do the job. I spent quite some time developing/optimizing a good solvent mixture.
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