Cd19 viogreen

Frozen PBMC were thawed in RPMI-50% FCS, counted in Trypan blue, and 1 × 10⁶ cells were stained with a viability dye (Viobility 405/520, Fixable Dye, Miltenyi-Biotech), Lineage antibodies (anti-CD3-BV510 (BD Biosciences), CD19-VioGreen (Miltenyi-Biotech) and CD56-BV510 (BioLegend)), and anti-CD14-BV650 (Biolegend), HLA-DR VioBlue (Miltenyi-Biotech), CD141-BV785 (BioLegend), CD16-PE (Beckman-Coulter), CD1c-BV605 (BD Biosciences), FcERI-PerCP-Vio700 (Miltenyi-Biotech), CD123-APC-Vio770 (Miltenyi-Biotech), CD11c-PE-Vio615 (Miltenyi-Biotech) antibodies (all antibodies are described in Additional file 1: Table S3). Samples were processed on the BD LSRFortessa cytometer (BD Biosciences). pDC and DC cells were identified as described by Mair et al. [49 (link)]. Data were analyzed using FlowJoTM version v10·8 software (BD Life Sciences).

Blood samples from the baseline visit were processed within 4 hours for analysis by flow cytometry. For lymphocyte phenotyping, ethylenediaminetetraacetic acid whole blood was stained for CD3, CD4, CD8, CD45, CD16, CD56, and CD19, as previously described (30 (link)). For B-cell phenotyping, PBMCs were isolated from lithium heparin whole blood by Ficoll gradient density centrifugation. One million PBMCs were incubated with the following antibodies: CD19-VioGreen, anti-IgD-VioBlue, CD24-PerCP-Vio700, CD38-FITC, CD27-APC, CD86-PE-Vio770, CD21-APC-Vio770, and anti-IgM-PE (Miltenyi Biotec, Bergisch Gladbach, Germany). Samples were measured using a FACSLyric flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data were analyzed using the FACSSuite (BD Biosciences). The gating strategy is shown in Supplemental Figure 1.