CM-H2DCFDA for Microscopy - (Aug/05/2009 )

Hi,

Does anyone have experience in using this dye for fluorescent microscopy? How are your results with it and what is your protocol for taking pictures?

I tried loading the dye and just visually analyzing the results under a fluorescent microscope. When excited by the lamp the cells fluoresces intensely for a brief momment (approx 5s) then the fluorescence dims over time. Fluorescence levels seems rather dynamic.

Product link http://products.invitrogen.com/ivgn/en/US/...=Search-Product

-Bill Nye-

flourescein compounds tend to be fairly unstable under light, sorry there isn't much that can be done about this. Make sure you use the right mounting medium and try putting a neutral density filter in the excitation path to dim the incoming light a bit.

-bob1-

Your observation seems about right. I also saw the signal fade within a relatively short amount of time because this dye photobleaches quickly. Make sure to use a mounting buffer with an antifade component (mostly antioxidants).

Can you analyze by Facs? That will get around the rapid photobleach.

-miBunny-

Thanks for the input! I forgot about the neutral filter, I'll give that a try... I'm just trying to see if I can get a general idea by first looking at the cells using fluorescent microscopy. There has been several publications with just fluorescent images using this dye so I thought I should at least look at the cells first before doing further analysis like FACS.

I also came across a pretty interesting article about these DCF dyes:
Afzel et al. Biochem BiophysRes Commun 304, 619 (2003)
"Most reports of experiments using DCFH as an ROS indicator show that concentrations at 5–50 μM were used. At such concentrations, however, DCFH photoreaction proceeds so rapidly that we consider that such concentrations of DCFH are especially unsuitable for fluorescence microscopic observation"

So far the above statement is holding true for me (I've been using 10μM for microscopy).

-Bill Nye-

Hi, Im new to using the CM-DCFDA assay for flow cytometry. I tried H2O2 treatment of a suspension cell line for 6 hours and checked unstimulated and stimulated cells. I cant get much of a difference between the samples. the reagent is new and just opened.
I treated the cells and then centrifuged them 2 x 10 5 cells per sample and added the probe 10uM in PBS. after 30 minutes I centrifuged the cells again and resuspended the cells in PBS again. AFter about 10 minutes incubation I checked FL1 log in flowcytometry. Unfortunately the NAC treated cells showed higher MFI than the H2O2 stimulated cells.
Im trying to trouble shoot the assy, if you have any suggestions please do let me know.
I wonder if the concentration of DMSO is too high but probably not. as the stock concentration of CM DCFDA was 1mM and I diluted in PBS from that stock.
can you suggest me some tips based on your experience with the assay?

-Chinthika-