Hi,
To study the relative amount of superoxide release according to different treatment, I use Mitosox and read the signal from the plate reader. Previously, I tried using the same conditions and looked through a microscope. But it was just hard to quantify.
Here is my experiment:
I treat the adherent cells with 5uM Mitosox in normal media (including phenol red) for 5 min in dark (foiled), 37C incubator. Then, I wash cells 2x with PBS (as instructed from the manual). Then I add PBS and take it to the plate reader. To read fluorescence, I use 5nm step and sum all the fluorescence from 565nm to 710nm (I have GFP in these cells, so I tried to avoid capturing the emission from it). I normalized the signal by the protein concentration from the cell lysate (from the same plate). Mitosox is diluted in DMSO from power just before using.
I am wondering if my conditions are okay. It takes too long to measure (perhaps 5nm step size is too narrow?). Do you suggest that I incubate cells with phenol red free media and also read the signal in the phenol red free media (with serum?)?
Thank you and have a great day.
Dear Eunjoo Lee
Greetings!
Try to avoid Phenol red and serum while staining.
You may make use of buffers like HBSS for staining.
Instead of a microplate reader if you can use flow cytometer it would be great as it is more sensitive.
I hope this suggestion works for your experiment.
Goodluck!
with regards
shubhankar
Thanks very much! I used a media without phenol red and read the fluorescence by microplate reader. Then I normalized the reading by protein concentration. At the time, I have access to only the microplate reader. Thanks very much Shubhankar and happy new year!
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