Facsdiva driven lsr 2 flow cytometer

Cells were stained with the following anti-human Abs: Phycoerythrin-conjugated anti-CD123 Ab (clone 6H6; BioLegend), allophycocyanin (APC)-conjugated anti–IFN-α Ab [clone 7N4–1; BD Pharmingen]. Flow cytometry acquisition was performed with a BD FACSDiva-driven BD LSR II flow cytometer (Becton Dickinson). Data were then analyzed with FlowJo software (TreeStar, Inc.).

Cytosolic hydrogen peroxide was measured using the Peroxyfluor-6 acetoxymethyl ester (PF6-AM) fluorescent probe as described in (Dickinson et al., 2011 (link)). In brief, cells were washed twice with warm PBS and incubated with 250 nM of PF6-AM in serum-free RPMI for 20 min at 37°C. Cells were allowed to recover in complete RPMI for 1 h and were subsequently harvested and resuspended in sorting buffer (PBS + 1% FBS). Flow cytometry acquisition was performed with BD FACSDiva^™-driven BD^™ LSR II flow cytometer (Becton, Dickinson and Company) which measured the increase in PF6-AM fluorescence. Data was analyzed with FlowJo software (Treestar Inc.)