To detect subpopulations and characterize phenotypes of B cells, PBMCs from SLE patients (n = 20) were separated and analyzed to determine the frequency of each B cell subset. Two × 105 cells were resuspended in 50 μl FACS buffer and incubated with surface antibodies for 15 min at 4 °C. Antibodies used included FITC-CD19, APC-CD21, APC/Fire750-CD27, Alexa/fluorro700-CD11c, PerCP/Cy5.5-CXCR5, PerCP/Cy5.5-IgG, PE/Cy7-IgD, APC-HLA-DR, PE/Cy5-CD69, and PE/Cy5-CD86 (Biolegend, San Diego, USA). After staining, cells were washed with 1 ml FACS buffer and resuspended in 300 μl FACS buffer for surface marker analyses.
The frequency and phenotype of DNA tetramer-binding B cells were detected by the tetramer staining technique, resuspended in 100 μl FACS buffer, and incubated with surface antibodies for 15 min at 4 °C. The stained B cells were washed with 1 ml FACS buffer and resuspended in 500 μl FACS buffer for flow cytometric surface marker analysis. FACS data were acquired with a BD FACS Canto II (Becton-Dickinson Immunocytometry Systems, San Jose, USA) and analyzed with FlowJo software (v. 10.0; Tree Star Inc., CA, USA).
The frequency and phenotype of DNA tetramer-binding B cells were detected by the tetramer staining technique, resuspended in 100 μl FACS buffer, and incubated with surface antibodies for 15 min at 4 °C. The stained B cells were washed with 1 ml FACS buffer and resuspended in 500 μl FACS buffer for flow cytometric surface marker analysis. FACS data were acquired with a BD FACS Canto II (Becton-Dickinson Immunocytometry Systems, San Jose, USA) and analyzed with FlowJo software (v. 10.0; Tree Star Inc., CA, USA).