I have been using CellROX Green to label my cells in a live cell fluorescent imaging format. My protocol is very simple:
-wash cells with PBS
-Add 5uM CellROX Green
-Immediately perform 45 minute time lapse imaging of cells (to include the 30 min incubation and 15 minutes post incubation in images and quantitative detection)
In comparing fluorescent ROS detection of my diseased and non diseased cells, I initially noticed stark and expected differences in my images. When I switched to a new tube in the same lot, and since then a completely different lot #, almost all my fluorescence is extremely low in every cell and I can't replicate the images I've previously obtained. The only time I see clear/vivid fluorescence is when I accidentally photo-oxidixed the dye with light, which isn't what I want.
I've seen other people post issues with CellROX Green and that it requires a lot of optimization. Has anyone else experienced difficulty with consistency and reproducability?
Thanks.
Try to ask Claus Dieter Schuh.
He just responded question related.
https://www.researchgate.net/post/DCF_staining_in_oocytes_for_oxidative_stress_Why_does_the_staining_intensity_continuously_increase_under_the_fluorescence_microscope#view=5886fa80eeae3920957aaad3
What is your positive control, and is it the same between both lots? One thing that isn't well-known about this dye is that H2O2 is not a good positive control for it. Also, PBS is probably not the best choice. I suggest something more physiological, such as HBSS, phenol red-free media, or FluoroBrite DMEM.
Thank you Daniil and Jason, I'll try a different buffer. I haven't used H2O2, and have been using Menadione and 1,4 napthiquinone as positive controls but will keep trying.
Check the concentration/time of menadione. We have used a treatment of 100 μM
menadione for 1hr at 37C. Oh, and be sure to use a fresh vial of the stock. An old stock may oxidize in the tube.
Just to add, try to buffer your media on the same incubator to be sure you don't shock the cells when washing/adding the dye. Had similar issues when working on oxidative stress in the past. Minimise disrupting the cells as much as possible.
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