Hi Everyone,
I have a query regarding C2C12 Cells, my Colleague did a DHE (ROS detection experiment) in which its layers get disturbed, She seeded 100,000 Cells P# 39, in 6 well plate.
And allowed them to get confluent for three days, She proceeded with Dihydro ethidium (DHE) on fourth day, but cells layer disturbed when incubated with DHE (10 uM working conc in PBS) for 30 min.
We don't know its exact reason, that either its incubation time with DHE which was 30 min, is long or cells suffering from starved condition, but can cells suffer from starved condition in this short 30 min incubation in DHE sol, can anyone please give a valuable comments.
Any discussion/ comments will be highly appreciated.
Thanks
Hello Shabbir,
the first problem seems to be cell density which is very high as 1lac cells/well of a 6-well plate and then a 3 days incubation. I am sure that the reason of cell layer getting disturbed is high cell density. You can seed 50,000 cells/well in case of fast growing cell lines and that works very well. Second, incubation time is 15-30 minutes for DHE and I dont think that, incubation time is a problem for cells to get deadhere. Third, I didn't get this thing that why you people are giving DHE treatment to a monolayer of cells. I think you should trypsinize the cells first and then give them a PBS washing and after that you can incubate them with DHE in required concentration and time to finally acquire in FACS machine.
Hope it helps..!
Thanks Ajay.
We did microscopy for monolayer cells, and for the next time, i will reduce the number of cells/ well as you recommended.
And i will also try to follow the same experiment in suspension after trypsinization, followed by FACS analysis,
did you have some sorts of experience the diffrence between the two outcomes. (i.e Through microscopy and FACS).
Anyway, i think this time it will work. Again Thanks for your comments.
Yes Shabbir,
Depends what type of results you require. For exact relative quantification, FACS is prefered over Microscopy because you cant get quantitative results in case of microscopy and only qualitative results are there. So it is really difficulte to point out relative differences between different cell types, while in case of FACS analysis, you get a quantitative value. SO FACS is better, but if you want fluorescent cell pictures also then you can do microscopy in parallel wells but FACS is necessary for quantitative results and most of the people report DHE in terms of FACS..
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