Previously I tried with EBM-2 (cell: HUVEC), but I found that in cell free condition EBM-2 in presence on DCFDA (20uM) gives very high fluorescence.
My plan is to replace EBM-2 with HBSS. But, I wonder which HBSS (with or without Ca2+/Mg2+ would be the appropriate choice?
Thanks in advance.
Dear Khan! I am sure that only a professional in this particular scientific field will be able to help you. By the way, please evaluate some papers on similar topic.
Vladimir
Hi Khan,
I will suggest you to try with the HBSS without the Ca/Mg. I am not sure about the EBM-2, but some cell lines grow poorly under Ca/Mg presence condition.
Arif,
You could make a blank with HNSS and the DCFDA, but without cells and discount the fluorescence.
@Dear Vladimir, you've been always a great contributor. I do appreciate your sincerity.
@Dear Wai, thank you for the answer. If this about growing, then I have to say my cells are well-grown there in the wells and I'll be keeping them in HBSS only for 30min (DCFDA) + 1hr (inducer + treatment). And after the wash (PBS/HBSS without Ca2+/Mg2+) there seems to be no significant detachment. So, concern was is there anything to do with DCFDA in the presence or absence of Ca2+/Mg2+ in the buffer, that can seriously affect the fluorescence intensity.
@Dear Marlize, I did that before commencing my experiment. I'll add more control of course. Thank you.
The dichlorodihydrofluorescein diacetate (DCFH-DA) dye has several limitations and it suffers interference from many sources.
Take a look at: Forman HJ, Augusto O, Brigelius-Flohé R, et al (2015) Even free radicals should follow some rules: A Guide to free radical research terminology and methodology. Free Radic Biol Med 78:233–235. dos: 10.1016/j.freeradbiomed.2014.10.504
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