Percp cy5.5 mouse anti human cd279 pd 1

Human fresh peripheral blood samples of all individuals were obtained from the children with acute HSP and HCs. Peripheral blood mononuclear cells (PBMCs) isolated by density gradient centrifugation with Ficoll-Hypaque solution (CL5020, CEDARLANE, Netherlands) were transferred to sterile tubes and washed twice with phosphate-buffered saline (PBS). Subsequently, human CD4⁺T cells were isolated by human CD4⁺T cell isolation kit (number 130-096-533) (Miltenyi Biotec GmbH, Germany). Additionally, human PBMCs were stained with FITC-Mouse Anti-Human CD4 (BD Biosciences, San Diego, CA, USA), clone name: RPA-T4, isotype control: FITC Mouse IgG2b, κ, clone name: eBMG2b; PE-Mouse Anti-Human CXCR5 Biotinylated Monoclonal Antibody (BioLegend, San Diego, CA), clone name: J252D4, isotype control: PE Mouse IgG2a, κ, clone name: eBM2a; APC-Anti-Human ICOS (CD278) (BD Biosciences, San Diego, CA, USA), clone name: ISA-3, isotype control: APC Mouse IgG2a, κ, clone name: MOPC-173; PerCP-Cy5.5 Mouse Anti-Human CD279 (PD-1) (BD Biosciences, San Diego, CA, USA), clone name: EH12.1, isotype control: PerCP-Cy5.5 Mouse IgG1, κ, clone name: MOPC-21. All the staining procedures were performed according to the manufacturers' protocols, and the stained cells were analyzed by a flow cytometer of FACSCalibur and CELLQUEST software (Becton Dickinson, Sparks, MD, USA).