Flow cytometry gating strategy.

A) Gate P3 was set to comprise all events with high CD14-FITC-fluorescence. B) Among events in P3, monocytes were included in gate P4 based on light scattering characteristics (FSC and SSC). C) Gate P1 was set to comprise lymphocytes based on light scattering characteristics, and for analysis all events in P1 but not in P4 were considered lymphocytes. Gate P2 was set to comprise neutrophils. pSTAT6-Alexa Fluor 647 and pSTAT1-Alexa Fluor 647 fluorescence intensity histograms were created for lymphocytes and monocytes in respective tubes. Representative histograms are shown for comparatively high (D) and low (E) monocyte pSTAT6 activation, and comparatively high (F) and low (G) lymphocyte pSTAT1 activation. Gate P5 (D, E) or P7 (F, G) is set to comprise 2–4% of events in the unstimulated sample and copied to the corresponding stimulated sample, i.e. the unstimulated samples serve as controls for the stimulated samples.