lucadandy

Hello!

I'm just a fresher of DCF Assay. I's need some help, advise with ROS measurement with my 3t3 cells as in the last months I've been getting some strange results.

experiment 1: (in 12 wells plates)
- 3t3 confluence
- DCF-DA 20 uM + phenol red free medium 30' covered by alu foil. (incubator)
- wash cells with pbs
- H2O2 0,1, 0,5, 1 mM in HBSS or PBS
- fluorescence reading with 485,520 filters  at 30', 1h, 2h, 4h,8h, 24h.

Experiment 2
3t3 confluence
- DCF-DA 20 uM + phenol red free medium 30' covered by alu foil. (incubator)
- wash cells with pbs
- H2O2 0,1, 0,5, 1 mM in HBSS or PBS + NAC 5 mM
- fluorescence reading with 485,520 filters at 30', 1h, 2h, 4h,8h, 24h.

my questions are as following.
1. is the [H2O2] too high? Should I use a lower concnetration
2. is the phen red medium good for DCF-DA
3. What about HBSS or PBS for 24 h treatment?
4. Would you use DCFDA before the H202 or would you stress the cells before with H2O2 and then reading the fluorescence after 30 minutes of DCFDA?

Let me know if is unclear.

Can't wait for your advise and suggestions.

thanks