Facs canto 3l

Cell viability was determined using L/D eFluor™ 450 (eBioscience, 65-0863-14) followed by surface antibody staining in FACS buffer. Cells were incubated with surface antibodies for 30 min in the dark. To detect CAR expression, cells were stained using goat anti-mouse IgG-biotin (Jackson ImmunoResearch) followed by streptavidin-PE or streptavidin- eFluor450 (ThermoFisher, 12-4317-87 and 48-4317-82). Intracellular staining was performed with the Foxp3/Transcription Factor Staining Buffer set (ThermoFisher, 00-5523-00) according to the manufacturer’s instructions. All experiments were performed on a FacsCanto 3 L, Fortessa 4 L HT and Fortessa 5 L (BD Biosciences) and the data was analyzed with FlowJo software (V.10, TreeStar). Antibodies listed on Supplementary Table 2 were used.

Neutrophil purity for in vitro migration assay and Adrb1 expression analysis was evaluated by incubating cells with Dylight-650-conjugated anti-1A8 Ly6G and with DAPI to assess viability. Mouse primary blood leukocytes from peritonitis experiments were incubated with anti-Gr1 conjugated with AF-647 and with PE-conjugated anti-CD115 and DAPI. Neutrophils were gated on the basis of Gr1-positive and CD115-negative staining in a FACS Canto-3L flow cytometre equipped with DIVA software (BD Biosciences). Doublet discrimination and viability (negative to DAPI) was assessed for every sample. Data were analysed with FlowJo (Ashland) software by blinded observer. All experiments were conducted at the CNIC-Cellomics Unit.