I want to measure growth inhibition using HCC1599, but as it is a suspension cell line, I need to remove the medium before I add the MTT solvent without losing cells.
Why you are not using MTS? you culture your cells in certain conditions, before the end of the experiment you add MTS wait for 2-5 h ( depend on cell type) then measure.
Add the MTT solution to the media, wait for the amount of time you want, transfer everything in a 50 ml tube, may be washing the culture vessel a couple of times with PBS, then spin down at the max speed allowed by the tube manufacturer, carefully dispose of the supernatant and then dissolve the formazan pellet in the amount of isopropanol of your choice. Transfer 200 micro-liters into the wells of a 96 well plate, in triplicate, and then measure the absorbance at 570 nm
Dear Mojtaba, MTS is a close derivate of MTT, the only difference is that it is metabolized in a reddish soluble products that dissolve in the media. The mechanism of action is exactly the same, so if you are using media with phenol red, you need to ave an expensive plate reader that does measure two different abs at two different wavelengths: one MTS+phenol red, one phenol red alone and then subtract the value of the second from the one. It is OK if you have the right equipment, otherwise the absorbance will be summed and you will need to do a lot of calculation to obtain MTS absorbabce alone.
I agree with Danilo and his explanation how to do MTT! Its the best/cheapest method!
Instead of isopropanol if you don't have it you can use DMSO...
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