we are working on different model system including leucocytes and cancer cell cultures to determine the intra-cellular ROS production.
And does cells incubation for 24hrs or 48hrs gives you better result or freshly isolated cells give out more ROS production?
Whatever the answer, don't use DCF. It is a very unreliable probe.
What is your actual goal? Would you be specific?
And DO NOT use DCFDA.... thanks David
Well Iam checking intracellular ROS generation of pbmc and cell lines in vitro. i want to see how much ROS can be produced by cells in suspension and adherent one. FOr control iam using H2O2 to induce ROS and while untreated ones are being checked for their ROS levels.
So, I reckon you are looking for the "condition" (e.g. starvation) which generates ROS.
In that case you need provide some more info...
# Incubation condition: serum free or % of FBS?
# DCFDA: concentration, time
H2O2 itself is a ROS, the term inducer does not fit to it.
#H2O2: concentration, how long
with DCFDA you really can't be sure that you are measuring H2O2.
And, last but not the least, the question you've asked... the actual answer is in it.
that's what are you working on. Its not really easy to tell which system will give "MORE" ROS comparing to the others. There are so many things will come along the way of interpreting the whole thing.
Anyway, good luck.
thank you Khan Md Taufiqul Arif , your answer was really helpful.
Yes we are optimising the H2o2 and DCFDA and hopefully will get better results.
Gourav Chandan Have you optimised your protocol? I am planing to incubate cells in H2O2 for 24 hours, how should I do? Should I keep cells in OPTI-MEM containing H2O2 for 24 hours? or Should I treat cells OPTI MEM containing H2O2 for a few hours, then refreshing medium with DMEM containing serum and harvesting 24 hours later? I am using HCT116.
Thanks,
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