Measurement of phosphorylated ERK1/2 expression and PAR formation by flow cytometry

Freshly isolated PBMCs were treated with H₂O₂ (500 µM), PMA (phorbol 12-myristate 13-acetate, Sigma-Aldrich) (50 ng/ml) or PBS at 37°C. In some experiments, PBMCs were pretreated with a MEK1/2 inhibitor (25 µM PD98059), a PARP-1 inhibitor (2 µM PJ34, Sigma-Aldrich), DMSO (0.05%), or medium before exposure to H₂O₂ or PBS. In additional experiments, freshly isolated NK cells were pretreated with PD98059 (25 µM), PJ34 (2 µM), DMSO (0.05%), or medium before exposure to monocytes in the presence or absence of PMA (50 nM) or PBS. The PBMCs and NK cells were then washed in cold PBS, fixed with cytofix/cytoperm (BD Biosciences, San Diego, CA) and resuspended in ice-cold methanol (Sigma-Aldrich) (final concentration 90%). After 10 min incubation on ice, the cells were washed in PBS with 5% BSA (Sigma Aldrich) and permeabilized with perm/wash (BD) before staining with mouse anti-phospho ERK1/2 (pT202/pY204) mAb (BD) or mouse anti-poly ADP-ribose mAb (BD) followed by incubation with Alexa fluor-488-conjugated goat anti-mouse (Invitrogen) Ab. Cells were then washed and analyzed for PAR accumulation or phosphorylated ERK1/2 expression by flow cytometry.