Anti igd pe cy7

Cells were mechanically homogenized, followed by staining for surface molecules using LIVE/DEAD fixable Aqua (Invitrogen, 1:1000 dilution) and anti-CD4 PE-Cy5 (RPA-T4, BD, 1:50 dilution), anti-CD3 PE-Cy7 (UCHT1, eBioscience, 1:50 dilution), anti-CD45RA V450 (H100, eBioscience, 1:100 dilution), anti-CXCR5 conjugated with Alexa Fluor 488 (RF8B2, eBioscience, 1:50 dilution), Alexa Fluor 647 (RF8B2, BD, 1:50 dilution), or biotin (RF8B2, BD Biosciencese, 1:50 dilution)/streptavidin APC-Cy7 (1:400 dilution), anti-PSGL-1 PE (KPL-1, BD, 1:50 dilution) or APC (FLEG, eBioscience, 1:50 dilution), anti-ICOS FITC (C398.4A, eBioscience, 1:50 dilution), anti-CCR7 FITC (150503, R&D Systems, 1:50 dilution), anti-CD62L FITC (DREG56, eBioscience, 1:50 dilution), anti-CXCR4 PE-Cy5 (12G5, eBioscience, 1:50 dilution), anti-CD200 APC (OX104, eBioscience, 1:50 dilution), anti-OX40 PE-Cy5 (ACT35, BD, 1:50 dilution), anti-PD-1 PE-Cy7 (EH12.1, BD, 1:50 dilution), anti-CXCR3 BV421(1C6/CXCR3, BD, 1:50), anti-IL-2RA PE (M-A251, BD, 1:25), anti-CD19 APC-Cy7 (SJ25C1, eBioscience, 1:50 dilution), anti-IgD PE-Cy7 (IA6-2, BD, 1:50 dilution), anti-CD38 V450 (HIT2, BD, 1:50 dilution), and anti-IL-10R PE (3F9, Biolegend, 1:50 dilution).

For human basophil sorting, cell suspensions were incubated at 4ºC with Fc-blocking reagent (Miltenyi Biotec) or blocking 2.4G2 mAb to FcγRIII/II (BD PharMingen) and then stained for 30 min with the following antibody combination: anti-CD45 AF700 (clone: HI30), anti-FcεRI FITC (clone: CRA1), antiCD123 PE (clone: 6H6), anti-HLA-DR PerCpCy5.5 (clone: L243), anti-CD19 eFluor450 (clone: HIB19), anti-IgD PE-Cy7 (clone: IA6–2). Basophils were sorted with a FACSAria II (BD Biosciences) after exclusion of dead cells through DAPI staining. The purity of cells sorted this way was consistently >95%.