Hi ! I'm Yu, and planning to analyze ROS assay with H2DCFDA(Thermo) on MSCs.
A lot of protocols instruct that treat H2DCFDA at specific concentration for 5~30 min, 20~30 min of recovery time with medium after removing the buffer, and then trypsinize, centrifuge, reconstitute and finally analyze with FACS.
However, I should deliver my samples to another lab, as we don't have equipment for FACS. It takes about 30 minutes to there. So, the main question is, can I fix this samples with 5 min of 4% PFA after centrifuge or in any other step?
Most of protocols do not mention about fixation, so I'm not sure whether it is just not mentioned or not recommended.
Thank you !
fixing should help in stabilizing your signal, but it will affect the cells as well. I would recommend using a plate reader. That will give a more realistic view of ROS in cells.
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