I am doing MTT assay on Hela cells. i keep the drug for 24 hours after cells have attached and become confluent in the 96 well format. After that i add MTT at 0.75mg/ml and incubate for 2.5 - 3 hours. Then i add DMSO to solubilize the formaxan crystals and take absorbance at 570nm and 630nm for background. I have done this for Geldanamycin, then plotted the value. I need to know the IC50 for this compound on HeLa cells ! please help!
I have plotted using both Linear and Non linear regression models. The R square values in both are aroun 0.95, and the values i am getting of IC50 is around 40uM. I have chosen the non linear model as the data points are on the graph as compared to a more skewed model in the linear fit where lower conc values are not well represented. I plan to repeat again with 24 and 48 hour incubation. the range i have chosen are from 50nM, 100nM, 250nM, 500nM,1uM, 5uM, 10uM, 25uM, 50uM, 75uM, 90uM. I cannot go beyond 90uM as my stock concentration is 9mM and i take 100uL per well of Media( without FBS). Please advise.
Bhaskar your way of calculating is all correct, but i would suggest increasing the points of concentration in between and having more periodic distribution and then you may find it fittable in even in linear equation.
for example- go with 50 nm , 100nm, 150nm, 250 nm, 500nm, 1um, 5um 10um, 20um, 30 um, 40 um, 50um 60um, 70um....till 90um.
@Vishal: It does not happen linearly, In most papers it is fitted using three or four paramter non linear regression models. The change that happens in lower concentrations is very less as compared to changes that take place at higher concentrations, even for the same fold increase decrease. Linear plots are very rare, and are often incorrect. Aneways, i need to find the IC50 value asap. Thanks for the suggestion. The curves are generally a plateau followed by a sharp decline.
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