Hi Sci-mates,

I've been studying oxidative stress on HL-60 and THP-1 cells. However, my cells are GFP-transfected. So, my group decided to use a probe called CellROX™ Deep Red (Thermo: Catalog number: C10422) to study it. I fallow the original protocol:

1.Treat the cells with the test compound or drug. -> I use PMA (10 ng/mL)

2.Add the CellROX® Reagent at a final concentration of 5 μM.

3.Incubate the cells for 30 minutes at 37°C.

4.Remove medium and wash the cells 3 times with PBS

I am reading it using the Flow cytometer using APC and APC-A700 (CellROX™ Deep Red -> Color: Far-Red. Excitation/Emission: 644/665).

But every time I read it, the Fluorescence Intensity is high, and all the cells (99%) are dyed. Even though when diluted it in 2.5 μM; 1.5 μM; 0.5 μM; and 0.1 μM and there is no difference of Fluorescence Intensity when compared the PMA stimulated group.

I know that both HL-60 and THP-1 cells when stimulated using PMA (10 ng/mL for 30 min, 37ºC), they activate the ROS process. I used the DHR protocol as my positive control on HL-60 WT and THP-1 WT, so it worked just fine.

That's why I know my PMA is working and my flow cytometer is working. However, I can not use the DHR protocol on my GFP-transfected cells because both emit a similar fluorescence signal, so we are trying the CellROX™ Deep Red instead.

Do you guys have any tips or similar protocols to help me to reduce the high fluorescence signal/dye pigmentation of my cells with and without PMA?

Thank you all!

Have a great day/night ahead.

Hugs!