I intend to do an apoptosis asay with HUVECs and had two questions Firstly what concentration of H2O2 do I use as a positive control without inducing substantial cell death I intend to measure caspase activity? Secondly how do I get the required concentration from 30% H2O2?
Dear Akshay,
The suggested concentration of H2O2 to establish a positive control of apoptosis in HUVEC cell is ~180uM (For further reading pl see the attached paper).
To get answer for the second part of your question please follow the calculation:
H2O2 Mol Wt =34g/mol
Density of the stock 30% soln=1.11g/ml
therefore vol of 100 g H2O2 30% sol=90.09ml
Therefore 1L (=1000ml) H2O2 sol contains (30g/90.09 ml)X1000 ml=333g
Therefore concentration of 30% stock=(333 g/l)/(34g/mol)=9.79mol/l=9.79M
Depending upon your final concentration you can serial dilute the stock to reach lower concentration. For making a bulk vol (1000ml) 180uM final dilution follow the v1c1=v2c2 general formula.
Here 9.79M=9.79X10^3mM=9.79X10^6uM
Therefore v1X(9.79X10^6uM)=(1000X10^3ul)X(180uM)
Therefore v1=18.38ul
Roughly you need to add 18.38ul of stock 30% H2O2 in 1Lit of water to get a final concentration of 180uM.
There is a very comprehensive answer in a parallel thread as well.
https://www.researchgate.net/post/How_to_prepare_40_mM_hydrogen_peroxide_from_30_H2O2_solution
Hope that helps!
H2O2 may not be the best positive control to induce apoptosis, for it can induce a very complicated kind of cell death including apoptosis, necrosis and others. I'm not sure whether you could using TNF + Smac-mimic to induce apoptosis. If that works, that cell death can be apoptosis for sure.
Christine Winterbourn, an expert on this topic has done detailed studies. You can find her studies on PubMed.
http://www.ncbi.nlm.nih.gov/pubmed/22229717
http://www.ncbi.nlm.nih.gov/pubmed/23713588
In general, doses of exogenous H2O2 below 200 uM is tolerable. Approaching 500 uM it becomes proapoptotic (still takes hours according to some works).
1 mM is kind of supraphysiological and perhaps unreal.
I have exactly the same question about H2O2 concentration to induce cell death but using rat ventricular neonatal cardiomyocytes. Can anyone help?
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