Hi, I am attempting to perform a reactive oxygen species (ROS) assay using the fluorescent probe CM-DCFH-DA (Invitrogen). I am using HaCaT human keratinocyte skin cells, and seed them at 40,000 cells/well in a black-sided, clear-bottom 96-well plate. After allowing the cells to adhere for 24 hours, I aspirate the media, wash with PBS, and add CM-DCFH-DA dissolved in 100% EtOH and 1% FBS media solution to the plate. That incubates for an hour at 37 C, 5% CO2 in the dark, and then I remove it, wash with PBS, and add various concentrations of H2O2 for an hour, and read the plate using a plate reader (485 ex/530 em). I am getting little to no fluorescence using this procedure. Any suggestions? Are HaCaT cells in particular difficult to penetrate with CM-DCFH-DA? Should I change my incubation times, etc?
Anna Carolina Paganini Guañabens
this actually appeared to my problem, and once I switched to non-phenol red media, it worked. Thanks a lot!
Hello. Should H2O2 be added to the plates prepared for the DCFH-DA protocol after 24 hours? Is reading done in flow cytometry 24 hours after adding H2O2?
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