Calcium dynamics via Orai1 supports GPCR-mediated PMN recruitment. PMNs isolated from murine Orai1+/+ (WT), or Orai1−/+ littermates, 2-day differentiated HL-60 cells, or freshly isolated human PMNs were loaded with fura-2 AM, perfused at a shear stress of 2 dyne/cm² into a microfluidic flow chamber consisting of an L-cell monolayer substrate expressing E-selectin, and then 0.1μM fMLP was perfused just after time 0 and ratio-imaged by fluorescence microscopy. (A) Representative images of a human PMN decelerating, arresting, and undergoing shape polarization show fura-2 emission, with the intensity of red representing 340 nm excitation (calcium bound) and green representing 380nM excitation (calcium unbound). The average calcium concentration is shown in 1-second increments, and the percentage of cells arrested is shown in 10-second increments. (B) Percentage of arrested and polarized in WT and Orai1−/+ PMNs is based on analysis of more than 150 cells from 4 paired experiments. WT cells had significantly higher arrest and polarization than Orai1−/+ at time points beyond 30 seconds. **P < .01. (C) HL-60 were transfected with negative control, or Orai1-specific siRNA as indicated, were differentiated for 2 days with 1.3% DMSO and were imaged in the flow channels responding to 0.1μM fMLP as in panel A (n = 10 separate experiments). Negative control transfectants were treated with 2-APB or U73122 to completely block SOCE or store release. For Ca²⁺ quantified at 60 seconds. *P < .05. **P < .01. (D) Human PMNs were observed in absence of inhibition (n = 6), in the presence of 100μM 2-APB (n = 4), or in the presence of 1μM U73122 (n = 4) as indicated. Cells treated with 2-APB had significantly lower arrest fraction at 30 seconds (P = .003) and significantly lower polarization between 50 seconds and 100 seconds (P values range from .007 to .045 compared with the controls). *P < .05; #P < .05 (1-tailed t test, P = .059 by 2-tailed t test).