Flou 4 am

Human or mouse bone marrow neutrophils were stained with 10⁻⁶ M of Flou-4 AM (Invitrogen, Eugene OR) in the presence of 4×10⁻³ M of probenecid (Invitrogen, Eugene OR) for 30 minutes at 37 °C. Neutrophils (5×10⁵) were aliquoted per tube in Hank's Balanced Salt Solution (HBSS) and maintained on ice and in the dark. Some samples were pretreated with 3×10⁻³ M EGTA, 10⁻⁵ M DPI, 10⁻⁵ M of PPQ-102 for 20 minutes or with 5×10⁻⁶ M of VX-770 for 30 minutes at 37 °C. The accumulation of intracellular free calcium was assessed by FACS in triplicate with a LSR II cytometer, using the blue laser (488nm) for excitation and 505 LP 530/30 BP filter channel for acquiring fluorescence emission. Neutrophils were collected before stimulation for 30 seconds at low acquisition rate to determine baseline calcium levels, then cells were stimulated with 10⁻⁷ M of IL-8 (R&D Systems), 10⁻⁷ M of platelet activating factor (PAF) (Sigma Aldrich), 10⁻⁷ M of C5a (R&D Systems) or (10⁻⁸ - 10⁻⁷ M) fMLP (Sigma Aldrich). The fluorescence emission of Fluo-4 was measured for 250–350 seconds. Data was analyzed using the kinetics platform in FlowJo software (Ashland, OR), each second of the kinetics represents the MFI of around 800 events.