P2X7 regulates phagocytosis of live or dead bacteria in vitro. Human PBMCs (2 × 107/mL in 150 μL of Na medium) were labeled with APC-conjugated anti-CD14 mAb and incubated with or without (A) 1mM ATP for 15 minutes, 0.3mM OxATP for 30 minutes, 20μM CytD for 30 minutes, or 50 μg/mL poly I for 30 minutes; or (B) 3 or 15 μg/mL P2X7-ED for 20 minutes at room temperature before the addition of 10 μL of CFSE-labeled live S aureus. After 60 minutes, cells were incubated with 100 μL of 0.25% trypsin for 2 minutes, followed by vigorous stirring and fixation. (C-D) Typical flow cytometry histograms showing phagocytosis of S aureus (C) and E coli (D) by HEK-293 cells transfected with P2X7-pCI constructs. Cells (3 × 106) were incubated with 20 μg of Alexa 488–conjugated bacteria in 150 μL of Na medium with 0.5mM Ca2+ for 5 hours (S aureus) or 10 hours (E coli). Cells were fixed, and fluorescence from surface-attached bacteria was quenched by trypan blue. Dotted lines show autofluorescence of transfected HEK-293 cells.
