I'm planning on looking at oxidative stress markers in quail red blood cells we have stored at -80. I'm planning to run them on an ELISA measuring protein carbonyls and SOD concentrations among other things.
I would like to know if anyone has managed to run similar techniques on frozen RBCs as opposed to freshly prepared blood cells and if they encountered any issues or offer advice on how to prepare them correctly.
Many thanks,
Dave
Dear David,
there is an established method to measure the grade of hemolysis in red cells. e.g. https://www.ncbi.nlm.nih.gov/pubmed/23822150
You stress them with various pro-oxidants (e.g. H2O2) and measure the rate of hemolysis.
SOD is also possible. Here two of our papers, where I guess the method is explained.
https://www.ncbi.nlm.nih.gov/pubmed/18981936
https://www.ncbi.nlm.nih.gov/pubmed/25527737
I hope this helps.
Good luck.
Karl-Heinz Wagner
Hello David,
i think that you should take a look at spectrophotometrycaly determinatios in protein carbonyls via DNPH and SOD activity via cytochrome C. Bothassays have great recovery, are relatively cheap and fast. If you want some assays about the aforementioned i would glad to attach you!
Hi David, I am working at the University fo Glasgow on Japanese quail too, with a main focus on oxidative stress. Please send me an email if you want more info, but for methods to measure oxidative stress from red blood cells, you can have a look at most of my papers on RG. I measured protein carbonyl, DNA damage, SOD, GPx, glutathione (total and oxidized).
All the best,
Antoine
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