I'm using abcams kit DCFDA/H2DCFDA CELLULAR ROS ASSAY KIT to measure ROS in human peripheral blood mononuclear cells by flow cytometry, but I'm having some trouble with positive control (TBHP), I have used different concentrations (50, 100 y 250uM) and negative control.
Hello,
I have no experience of the DCFDA assay, but my guess is that your PBMCs aren't cleansed off of Ficoll, which may interrupt NADPH production, which is essential for TBHP to work. (https://www.google.co.jp/amp/s/www.researchgate.net/figure/Diminished-intracellular-NADPH-oxidase-derived-ROS-production-in-Ficoll-Paque-enriched_fig3_333668825/amp). That is, if you are using Ficoll as your isolation method.
Myeloid cells are the primary source of ROS. Neutrophils and to a lesser extent monocytes express ROS. Typical ways to activate myeloid cells includes 30 ng/ml PMA often with 2 uM ionomycin or with 1 ug/ml LPS.