I am measuring the ROS and my drug activity in ROS. I dont understand how can i analyze my results. Under the fluorescence microscope i got images. But i dont know how to analyze them through the fluorescence micro-plate reader and flow cytometry. I can get some through the machine but how to analyze or calculate the results.
Please explain it. I would be grateful.
Thanks
I used flow cytometry to analyze DCFDA. The brightness of DCFDA can be calculated by mean fluoresce index (Geom. Mean) in flowjo. It the cell size is different among groups, the MFI can be normalized by FSC value.
Hi Jahan,
If your cells generate more ROS, expect increased GFP signal (compared to you control cells)
DCFH-DA will be converted DCFH by esterase, after that depending on the ROS level DCFH will be converted to DCF (which gives you fluorescence)
A) Using F-microplate reader compare values that you got from 530nM (compare control and treatment group) also you need to normalize with viable cells (MTT assay: in viable cells will produce more formazan (which will be measured at 570nm))
example:
control group 530 value: 3 (lets say) and 570: 2
treated group 530 value: 1 and 570: 2
now you can calculate:
control 3/2=1.5
treatment 1/2=0.5
based on that your treatment somehow attenuate production of ROS (or ROS consumption increased)
B) As Guo suggested, using MFI you will be able to say if you got more/less fluorescence in you groups. (bases same only way you detect fluorescence is different)
Goodluck,
Thank you so much. I just attached my result. I am new at this flow cytometry. Could you please explain it that would be great. Thanks
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