I was trying to detect H2O2 induced intracellular Reactive Oxygen Species (iROS) generation with DCF-DA fluorescent dye (2′,7′-Dichlorofluorescin diacetate, Sigma Aldrich, St. Louis, MO) in HK-2 cells. For my surprise, I was observing higher fluorescence values (using a plate reader, Ex 485nm cut off 530nm, Em 538nm) for the control groups verses H2O2 treated groups. Does anyone have an idea, what’s going on?

Thanks,

Susara

Protocol briefly:

Seeding 10 000 cells/well in a black 96 well plate, 24h incubation, H2O2 treatment 0.1, 0.2, 0.3, 0.6 and 1mM for 1, 2, 3 and 4 h. Cells were washed twice with HBSS and 10uM of DCF-DA dye in HBSS was added and incubated for 30min.

The dye was excited at 485nm and the emission was recorded at 538nm.

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