I have been trying to do MTT assay using HCT-116 cells but I have been getting very low absorbance readings (absorbance is usually 0.25 and below) even though iam seeding 30,000 cell/ well. I have been passaging the cells for around 18 generation now. I have noticed that the morphology of the cells under microscope have changed but they are growing normally. Also Iam getting gradually decreasing absorbance for the first 5 concentrations but then it increases again in the second 5 concentrations. the graph now is V shaped. what can be the reason for these irregularities?
If your cells are not doubling normally then that is the problem.
If the untreated control and the vehicle control are not showing the highest and the lowest absorbance values in the dataset respectievly, then the problem is with your MTT reagent or the formazan solvent.
If both the situations are not your case, then the more likely possiblity could be the drug you are studying. Some compounds that are anti-oxidants or drug efflux pump inhibitors can interfere with the MTT assay. You may need to assay toxicity of these compounds by alternate means.
Reference :
Article Drugs with anti-oxidant properties can interfere with cell v...
Praveesh Valissery
But my other colleagues are using the same MTT aliquots and reagents but they have no problem. as for the drug I found several papers about MTT assay for it. my colleagues also are working with it with no problems. The cells are growing fast actually; I think faster than their initial rate when i started passaging them. I was thinking that maybe prolonged passaging could have altered their metabolic activity so they might be less efficient in degrading MTT. Is this a possibility?
Yeah, it is a possiblity. There are reports of older cultures (>30 passages) showing reduced mitochondrial DNA. People generally stop using cell lines that have exceeded 20 passages. You can use a younger line and compare with the current passage to be sure.
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