Analyzing FPR2 Signaling in HL60 Cells

HL60 cells stably transfected with human FPR2/ALX (HL60-FPR2) have been described recently (23 (link)). These cells were grown in RPMI medium (Biochrom) supplemented with 10% FCS (Sigma-Aldrich), 20 mM Hepes (Biochrom), penicillin (100 units/ml), streptomycin (100 µg/ml) (Gibco), 1 × Glutamax (Gibco) and in the presence of G418 (Biochrom) at a final concentration of 1 mg/ml. Neutrophils or HL60-FPR2 cells were stimulated with different bacterial culture filtrates (USA300, USA300Δαβδ, USA300ΔeapΔH1ΔH2::ermB, S. epidermidis RN1457) or FPR2 ligands (PSMα3, PSMε) as described in the figure legends. Calcium fluxes were analyzed by stimulating cells loaded with Fluo-3-AM (Molecular Probes) and monitoring fluorescence with a FACSCalibur or a FACS Fortessa flow cytometer (BD Biosciences) as described recently (23 (link)).