Both anti-P2X7 mAb (L4) and recombinant P2X7-ED inhibit phagocytosis of YG beads and bacteria. (A) A representative flow cytometry dot plot showing the inhibitory effect of L4 mAb on bead uptake. Human PBMCs prelabeled with APC-conjugated anti-CD14 mAb (2 × 106 in 100 μL) were incubated with L4 or the isotype control mAb WMD7 at indicated concentrations for 30 minutes at 20°C before dilution to 1.0 mL and the addition of 5-μL YG beads. The fluorescence from bead uptake was measured in gated CD14+ monocytes. The dose-response curve is shown in panel B (n = 3 or 4). (C) A representative flow cytometry histogram showing the inhibitory effect of L4 F(ab′)2 fragments on phagocytosis of E coli. Human PBMCs (3 × 106 in 150 μL) were incubated with L4 or WMD7 F(ab′)2 fragments at indicated concentrations for 30 minutes at 20°C before the addition of 20 μg of Alexa 488–conjugated E coli (10 μL). After a 60-minute incubation at 37°C with gentle shaking, cells were fixed and the fluorescence was measured immediately after the addition of trypan blue. (D-E) A total of 5 μL of YG beads was incubated in Na medium with or without P2X7-ED at indicated concentrations (μg/mL) in a total of 30 μL volume for 10 minutes. Heat-treated P2X7-ED (95°C for 10 minutes) was used as a control. The mixture was added into 1 mL of fresh human mononuclear cells (D) or THP-1 cells pretreated with or without 20μM CytD (E). (F) A total of 10 μL of Alexa 488–conjugated S aureus was incubated in Na medium with or without 52 μg/mL P2X7-ED in a total of 30 μL volume for 10 minutes. Heat-treated P2X7-ED was used as a control. A total of 130 μL of cells (∼ 3 × 106) pretreated with or without 20μM CytD was added. The mixture was incubated at 37°C with gentle shaking for 20 minutes, and stopped by 150 μL of 4% paraformaldehyde. Equal volume of 1% trypan blue was added immediately before analysis by flow cytometry.
