I'm trying to do an experiment where I injure neurons with H2O2 and apply that injured neuron medium to glial cells. For this purpose, I'll injure neurons for 30min and then freeze the medium until use on glial cells (when i re-warm to 37C and use). Any insights into how the H2O2 concentration in the medium might fare after the freeze-thaw cycle? Just to ensure that I study the resultant effects correctly in terms of neuron-induced effects Vs remnant H2O2-induced effects in these glial cells. Thanks in advance!
Thanks Vamsi. I will do that, hopefully should have some useful endpoints to compare directly this way.
Depending on the initial concentration of H2O2 and media type during neuronal treatment, the H2O2 may be basically gone before freezing. H2O2 is metabolized by intracellular peroxidases and can reduce extracellular H2O2 rather quickly (about 95+% metabolized in 1 hr). Also if the media contains pyruvate this will promote scavenging.
http://www.jneurosci.org/content/17/23/9060.full.pdf
http://www.ncbi.nlm.nih.gov/pubmed/23485584
If you have managed to survive H2O2 decomposition by freeze media containing H2O2, then the exogenous H2O2 will need to deal with the components present in the media, pH, and 37C heating.
The best way to make sure you don't have a problem is to test the media for H2O2 concentration. You could use a luminol/hypochlorite assay or pHPA depending on instrumentation or amplex red.
http://www.sciencedirect.com/science/article/pii/S2213231713000323
Thanks John for your very detailed answer, it is very informative! I am treating neurons for 30mins with 50uM so am anticipating some H2O2 to remain in the medium but am hoping some degradation for sure with the entire freeze-thaw procedure plus the fact that I do have pyruvate in the medium. I'll try and measure the H2O2 content in the medium as u suggest, I have both these assays in the lab.
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