Transfection with P2X₇ confers phagocytic ability. (A) Bright field and fluorescent images showing engulfed nonfluorescent 3-μm beads within a monolayer of HEK-293 cells cotransfected with both NMMHC-IIA-AcGFP and P2X₇-DsRed. Cells were counterstained with 5μM 4,6-diamidino-2-phenylindole for 30 minutes and washed 3 times during a 1-hour period before incubation with 10 μL of beads for 10 minutes at 23°C. The images were captured by an Olympus IX81 fluorescent microscope with a 60× oil-immersion lens. (B) Phagocytosis of YG beads by HEK-293 cells transfected with P2X₇-DsRed or NMMHC-IIA-DsRed. Time-resolved flow cytometry dot plots allowed cells to be gated and analyzed as 2 populations according to their DsRed intensity (high or low). Control cells were transfected with DsRed alone. (C) Quantitation of YG bead phagocytosis by HEK-293 cells transfected with DsRed (mock) or wild-type P2X₇-DsRed. Cells were pretreated with IgG_2b isotype control mAb (clone WMD7, 100 μg/mL) and anti-P2X₇ mAb (clone L4, 100 μg/mL). Additional control was CytD added 20 minutes before the YG beads. Phagocytosis index of untreated cells (Basal) in each separate experiment (n = 3-5) was used to calculate the basal level (100%).