Dear RG community,
I have a substance that I want to use it in the cell culture (in vitro). I am using flow cytometry for mitchondrial dynamics and western blotting for mitophagy/autophagy. But I could not decide what dose I should use. The substance is FDA proved and does not harm to human. I started 100uM for some paper suggested (experiment details are all different than mine). I went up to 1mM and the effect that I am getting from flow cytometry is increased gradually but I do not know whether the dose is good or should I go up like 10mM or more? What is the limit and how to determine?
I am planning to do MTT assay 4h 16h 24h 48h 50uM 100uM 500uM 1mM 5mM and 10mM to see the cells growth. But MTT is different experiment and I am investigating different thing. How to combine them to choose proper dose. (By the way what time is more appropriate for MTT?).
I am master student therefore my knowledge is limited please let me know different experiments or protocols to continue. Thanks in advance.
Best
Süleyman
Hi,
First, the compound causes any cell death..? if so, determine it's IC50 value by doing FACS with PI. or you can simply do trypan blue staining.
when you see cell death(100uM), go for very less dose 2, 5, 10uM
Yes, you need to play with different dose levels to standardize
Good luck
You have to find the dose in which 50% of your cells are dead. That means you have to test different doses until you get the number you are looking for. I know it is a heavy job but science is more about repeating boring steps than inspiration.
If the drug is FDA approved you will find all the information at the US patent office.
Hi,
As they said, we should find the IC50.
By your MTT assay design you will be able to calculate it. I suggest you to fix the time, such as 24h and use the range of concentration. And then see if the IC50 is inside that range. To do so, you will use the MTT results comparing the drug and control results. You will find excel tables with models to do it.
Good luck ;)
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