Hi, I'm trying to study the impact of reactive oxygen species on whole blood with respect to how it may induce proteolysis on an intracellular protein in PBMC's in a simulated extracorporeal circulatory model. Instead of exposure of PMBC's to hydrogen peroxide, I need to mimic the actual inflammatory environment such as cardiopulmonary bypass and therefore need whole blood. Trialed H2O2 diluted down to 0.05% without much success. Any suggestions would be much appreciated.
It is not clear from your given details that which reactive oxygen species your are targeting for? For more focused suggestions, could you please make it bit more clear on your experimental targets? Anyway, with your given information, let me tell you that the hydrogen peroxide (H2O2) is a strong oxidizing agent and can generate OH- (hydroxyl ion) in a cell or solution. There are several reactive oxygen species and every one has its own pathway for damaging cellular histo-archtecture depending on the extent and nature of insult on the cellular/sub-cellular and macro-molecules like protein and lipids. You can measure H2O2 level in blood serum/plasma using sensitive kits from R&D systems, USA. Total NO levels can also be measured accurately by using Nitrate/Nitrite kits in blood samples using kits from R&D systems, USA and other company make kits. Thus, you can analyze both H202 and total NO levels in blood samples.
Thank you Shail K Chaube for your detailed answer. I was hoping for a recommendation for which ROS to use. My intention is to study the impact of exposure of whole blood to one of the ROS, such as H2O2, in a modified extracorporeal system, rather than measuring the production of ROS. I hope that clarifies my question? Thank you!
Hi Deyin, when we want to study a low-mild H2O2 environment, we usually use a Glucose-Glucose oxidase combination that can produce a low and consistent level of H2O2. For cell culture, 3mU/ml of glucose oxidase for 1 hour (in the presence of high glucose 500mg/L) can significantly stimulate H2O2-induced protein carbonylation, which is also better and more consistent than a bolus of high H2O2. Not sure if it will work under your condition dealing with PBMC.
I used to obtain a good oxidative shock by using cumene hydroperoxide at a concentration of 30-100 µM, within 60-120 minites
Please also try 3Morpholinosydnonomone SIN-1, Which is a potent ONOO- Generator, which induces not only oxidative but also nitrative stress simultaneously. Good luck with your research. Thanks.
Respectfully submitted
Dean Sharma
I would like to add that please do try DCFDA assay for total ROS measurement.
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