Anti cxcr5 bb515 clone rf8b2

PBMCs were thawed quickly, resuspended in complete medium in the presence of DNase I (10 U/ml, Sigma-Aldrich), and rested at 1 × 10⁶ cells/well in 96-well U-bottom plates (Corning) for 3 h at 37°C. For surface analyses, the medium was supplemented with anti-CXCR5–BB515 (clone RF8B2, BD Biosciences) and unconjugated anti-CD40 (clone HB14, Miltenyi Biotec), followed 15 min later by the relevant peptides (each at 0.5 μg/ml). Cells were then incubated for 12 h at 37°C. For intracellular analyses, the medium was supplemented with anti-CXCR5–BB515 (clone RF8B2, BD Biosciences), followed 15 min later by the relevant peptides (each at 0.5 μg/ml) and a further 1 h later by brefeldin A (1 μg/ml, Sigma-Aldrich) and monensin (0.7 μg/ml, BD Biosciences). Cells were then incubated for 9 h at 37°C. Negative control wells lacked peptides and contained volume-equivalent DMSO.