Hello,
I am working on inducing oxidative stress in cardiomyocytes using H2O2. I need to dilute the H2O2 stock to my required concentration. I use DMEM as the medium for cell culture. Do I need to dilute H2O2 in DMEM or dilute it in DMSO and treat the cardiomyocytes. Any suggestions are invaluable .Thankyou
it depends on the final concentration you want, you may dilute before in order to use bigger volumes when you treat the cells reducing errors and having more reproducibility. But you don't need DMSO for H2O2 dilution, you can use DMEM. (if you use DMSO you need to add the same volume in non treated cells as vehicle)
Is useful have a solution which is 1000X the final concentration required and you ad 1 uL per mL of medium
Having done this many times in our lab on cardiomyocytes, we dilute the H2O2 in PBS to a 20X stock, in order to add a larger volume to the cells. i.e. 500ul of 20X stock to 10ml culture medium. Addition to the cells is done dropwise throughout the petri dish followed by swirling the dish to mix the hydrogen peroxide into the medium. We do this to avoid the local build up of a high concentration of hydrogen peroxide at the addition site, which can have confounding effects and it makes our results much more reproducible.. Hope it helps.
Hi there, I routinely use H2O2 on my cells (cultured astrocytes/microglia) and I dilute it in DMEM without serum. While I grow my cells with serum, I use serum free for H2O2 or any treatments as serum can have catalase in it which can affect H2O2 treatment. I do my assays in 24/12 well plates so i make up my required dilution(s) of H2O2 in DMEM and add 500uL or 1000uL into each well. Hope this helps
Thanks a lot for your replies. I am using SOD2 as a marker for oxidative stress. I treat cardiomyocytes with 150μM H2O2 for 12 hrs. Is using the SOD2 as a marker a good option as some papers mentioned that SOD2 degrades as the stress increases. What is the best marker for oxidative stress?., thank you.,
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