I want to measure ROS synthesis in cells grown in transwell. I cannot measure fluorescence directly in the transwell because of the high so I have to remove the cells. One option is to use trypsin but I guess if someone has used another method. I use DCFDA kit (Abcam). Thank you
If possible attach the cells on the coverslip. Once the cells are adhered on the cover slip, wash the cover slip three times and then expose to H2DCF (10 micro Mol) for 30 min in culture medium. After washing three times with culture medium, cover slip can be observed under epifluorescence microscope
using excitation at 480 nm, emission at 520 nm. The fluorescence intensity reflects the total ROS level in a cultured cell. You can perform the corrected total cell fluorescence (CTCF) analysis following the method published earlier using
Image J Software to analyze the total ROS level.
Let me clear you that this procedure will assess the total ROS level in a cell but not ROS synthesis. Good luck
See the links here
Hope help you
Good Luck
http://mcms.unife.it/it/ricerca-1/ambiti-di-ricerca/patologia-oncologia-e-biologia-sperimentale/signaltransductionlab/publications/143.pdf
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4319729/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4031511/
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