Percp cy5.5 conjugated anti cd19

Isolated liver leukocytes or PBMCs were blocked with 5 μg/mL anti-mouse CD16/CD32 antibodies (BD Bioscience) and stained with fluorochrome-conjugated antibodies for 30 min at 4 °C in the dark. The cells were washed twice with PBS containing 3% FBS and fixed in 0.5 mL of Fluorofix buffer (BioLegend) for 30 min. The fixed cells were washed with PBS containing 3% FBS once and then analyzed on a FACSFortessa instrument (BD Bioscience). The fluorochrome-conjugated antibodies used in this study included FITC-conjugated anti-CD3, FITC-conjugated anti-CD11b, phycoerythrin-conjugated anti-CD45, APC-conjugated anti-CD8, APC-conjugated anti-CD115, APC/Cy7-conjugated anti-CD45, APC/Cy7-conjugated anti-Ly6G, BV605-conjugated anti-CD4, BV605-conjugated anti-Ly6G, BV421-conjugated anti-Ly6C, PerCP/Cy5.5-conjugated anti-CD19, PerCP/Cy5.5-conjugated anti-F4/80 (all purchased from BioLegend) and BV421-conjugated anti-CD11a (BD Bioscience). The compensation settings of the flow cytometer were determined with single-stained Ultracomp eBeads (Thermo Fisher Scientific). The frequency of each cell subpopulation obtained by flow cytometric analysis was further multiplied by the total isolated cell count to obtain the cell number for each subpopulation.

We isolated peripheral blood mononuclear cells (PBMCs) from the blood samples using density-gradient centrifugation on Ficoll-Paque. Single-cell suspensions were treated with fixable viability stain 510 (BD Bioscience, 564406) for half an hour, followed by staining with the following antibodies to distinguish the Tfh subsets: FITC- conjugated anti-CD45RA (BioLegend, N418), PE-conjugated anti-CCR6 (BioLegend, G034E3), PerCP/Cy5.5-conjugated anti-CXCR5 (BioLegend, J252D4), PE/Cy7-conjugated anti-CXCR3 (BioLegend, G025H7), APC-conjugated anti-PD-1 (BioLegend, EH12.2H7), and APC/Cy7- conjugated anti-CD4 (BioLegend, RPA-T4). For the detection of circulating plasma cells, PBMCs were stained with FITC-conjugated CD27 (BioLegend, M-T271), PE-conjugated IgD (BioLegend, W18340F), PerCP/Cy5.5-conjugated anti-CD19 (BioLegend, HIB19), and APC-conjugated CD38 (BioLegend, HB-7), followed by the treatment with fixable viability stain 510. Cell acquisition was performed using a CantoII cytometer (BD Bioscience). Data were analyzed with Diva (BD) software.