I treated pertussis cells with ethanol and then I analyzed them in a flow cytometer after staining with cDCFDA. I would like to know why I'm getting such a strong fluorescence signal with cDCFDA if the cells are already dead. Is this an artifact? I don't understand the reaction in this case.
Thank you,
Can you give additional info on your staining protocol? Usually DCFDA is used in living cell with a time of incubation that can vary with the cell type, with the kind of treatment you are using etc... Important is the medium in wich you load the cells with DCFDA. Finally depending on the exposure time with your molecule you can consider to load the cells with DCFDA before exposure. Regards
I'm just killing the cells with 70% ethanol, washing with 1xPBS, re-suspension in 1xPBS and adding cDCFDA (40microM), incubation time 45 min at ambient temperature. I'm doing this just to see what happens with dead cell. I usually use cDCFDA with live cells. Thank you.
Dear Vanessa, DCFDA is indeed recommended to stain viable cells. With fixed cells you obtain lots of false positive staining.
Regards,
Maurizio
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