I treat mesenchymal stem cells with 30 % of stock solution of H202. I want to use H2O2 as an apoptotic agent. Beside I would like to know exactly how much to use of the concentration to check the LC50 value. I will verify apoptosis by staining Dico6(3)/ Propidium iodide with flow cytometry. Thanks for your help!
You will need to test several concentrations. Maybe start from 100uM to 1000uM. Be sure to make the H2O2 fresh each time. Some of these concentrations may induce senescence whilst others will induce cell death.
Hi Marylin,
I am working with hydrogen peroxide and mesenchymal stem cells. I am agreed with Mr Dominick.
You can try with various concentration of H2O2 and you have to standardise the concentration.
Start from very low concentration to high concentration. You can start with 0,5,10,25,50,100,1000 uM. By the MTT assay, you will get the dose dependent % of survival of the cell. If the survival % is decreasing from 50-100 uM concentration, in the 2nd set of experiment, you have to take concentrations between 50-100uM (it may be 0, 50, 55,60,65,....... 10um). You will get LC50 Value also.
After standardisation the dose you can treat again with the specific dose of your interest (if it is 75uM take 5uM and 100uM too as +ve and -ve controls).
Then stain with Dico6(3) vs PI and analyse by using flow cytometry.
Many thanks for answering my question.
Your answers helped me very much. Thanks again.
Dear Marylin Mora: I am also working in H2O2 recently. I have checked different concentrations of H2O2 with hMSC cells. percentages of H2O2 0.000534058, 0.000267029, 0.000133514 seems to be not harmful to cells.
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