Calcium flux in suspension assay

Calcium flux assays were performed as described previously (36). Isolated neutrophils were suspended at 5 × 10⁶ cells/ml in RPMI/5% FCS/5 mM Hepes (pH7.4) then incubated with 5 μM fluo-3 (Molecular Probes, Eugene, OR) in the presence of F-127 detergent (0.02% -- Molecular Probes) in the dark at 37°C for 30 min with intermittent mixing every 5 min. The cells were washed twice with Ca²⁺- and Mg²⁺-free HBSS supplemented with 20 mM Hepes (pH 7.4) then diluted to 5×10⁶ cells/ml and kept at 4°C in the dark until used. Prior to analysis, the cells were aliquoted into FACS tubes, warmed to RT, then analyzed by flow cytometry (FACScan, Becton Dickenson, San Jose, CA) for 40 seconds to establish a baseline reading. Chemokines were then added and the samples analyzed continuously for 3 min. The data were analyzed by averaging fluorescence per 20 second intervals. Peak Ca²⁺ flux values were calculated by subtracting baseline readings.