Cxcr5 apc

Single-cell suspensions were prepared from fresh spleen tissue and followed by lysis of red blood cell (RBC) using ACK lysis buffer. For surface staining, 1 × 10⁶ splencytes per 100 μl were incubated for 30 min at 4 °C with the following fluorescently labeled conjugated mAbs: CD4-FITC (eBioscience, San Diego, CA), CXCR5-PE (eBioscience), CXCR5-APC (eBioscience), ICOS-PE-Cy5.5 (eBioscience), OX40-APC (eBioscience), CD40L-APC (eBioscience), PD-1-APC (eBioscience), CD19-APC (eBioscience), IL-21R-PE (BioLegend). After staining of surface markers, the cells were permeabilized with fixation/permeabilization solution (eBioscience) and subsequently intracellularly stained with Bcl-6-PE (BD Pharmingrn, San Jose, CA), the cells were then wash twice using permeabilization buffer, and eventually fixed with 4% paraformaldehyde in PBS. For APC-conjugated anti-IL-21(BD Pharmingrn), spleen cells were stimulated with PMA and Ionomycin for five hours and subsequently intracellularly stained, all procedures were in accordance with Bcl-6 staining method. In addition, Tfh (CD4⁺CXCR5⁺) subsets and B (CD19⁺) subsets were sorted with a FACS Aria cell sorter (BD Bioscience).

Phenotypic analyses of T cells and B cells were performed with anti-human monoclonal antibodies (mAbs): anti-human CD3-PerCP, CD4-FITC, CD19-PerCP and CD21-APC were from BD Biosciences (San Jose, CA, USA). CXCR5-APC, ICOS-PE, PD-1-PE, IFN-γ-PE, IL-4-PE, IL-17-PE, IL-21-PE, IL-22-PE, CD27-FITC, CD86-PE, CD95-PE, CD25-APC, and Foxp3-PE antibodies were obtained from eBiosciences (San Diego, CA, USA). Cells were analyzed by flow cytometry (BD FACSCalibur, San Jose, CA) and data was analyzed by FlowJo software (Tree Star, San Carlos, CA).

To determine the NK cells and lymphocyte subsets in patients, heparin‐anticoagulated whole blood samples were collected and stained with (1) CD3‐PerCP (BD Biosciences, San Jose, CA,), CD4‐BV510 (BD Biosciences, San Jose, CA), CD8‐APC (BD Biosciences, San Jose, CA), CD45RO‐BV421 (BioLegend, San Diego, CA), and CCR7‐PE (BD Biosciences, San Jose, CA); (2) CD4‐APC‐Cy7 (eBioscience, San Diego, CA), CXCR5‐APC (eBioscience, San Diego, CA), TIM‐3‐PerCP (eBioscience, San Diego, CA), TIGIT‐FITC (eBioscience, San Diego, CA), CD226‐PE‐Cy7 (eBioscience, San Diego, CA), PD‐1‐BV510 (BD Biosciences, San Jose, CA), and ICOS‐BV421 (BD Biosciences, San Jose, CA); (3) CD19‐PE (eBioscience, San Diego, CA), CD27‐PE‐Cy7 (eBioscience, San Diego, CA), CD38‐APC (eBioscience, San Diego, CA), CD24‐APC‐Cy7 (eBioscience, San Diego, CA), IgM‐BV421 (eBioscience, San Diego, CA,), and IgD‐FITC (eBioscience, San Diego, CA); and (4) CD3‐FITC (BD Biosciences, San Jose, CA) and CD16 + CD56‐PE (BD Biosciences, San Jose, CA). Cell counting was performed with absolute counting tubes with a certain number of beads (BD Biosciences, San Jose, CA). The samples were measured using FACS Canto II (BD Biosciences, San Jose, CA). Gating analysis was performed with FlowJo V10 (BD Biosciences, San Jose, CA).