Cxcr5 rf8b2

Patients’ and healthy control (HC) PBMCs were purified by Ficoll density gradient centrifugation, washed with RPMI 1640 plus penicillin, streptomycin, and L-glutamine along with 10% FBS (R10) and filtered through a 40 µM strainer. Cells were resuspended to a concentration of 1 × 10⁶ cells/mL and aliquoted (1 mL/well) into 48-well plates. PMA (final concentration 20 ng/mL), ionomycin (1 µM) and brefeldin A (5 µg/mL, Sigma-Aldrich) were added prior to incubation at 37°C for 5–6 hours. After incubation, cells were washed with FACS buffer and stained with LIVE/DEAD Blue (Life Technologies). Intracellular staining was performed using BD CytoFix/CytoPerm (BD Biosciences) reagents according to the manufacturer’s instructions using the following antibodies: CD3 (SK7, BD Biosciences), CD4 (RPA-T4, BD Biosciences), CD8 (SK1, BD Biosciences), CD45RO (UCHL1, Beckman Coulter), CXCR5 (RF8B2, BD Biosciences), CD25 (M-A251, BD Biosciences), IL-2 (MQ1–17H12, BioLegend), IL-4 (8D4–8, BD Biosciences), IL-5 (JES1–39D10, BD Biosciences), IL-13 (JES10-5A2, BD Biosciences), IFNγ (B27, BD Biosciences), IL-17A (eBio64DEC17, eBioscience). Cells were gated on viable CD3⁺ CD4⁺ CD45RO⁺ cells. Ex-vivo Treg staining was performed using anti-CD3 (UCHT1), CD4 (OKT4), CD25 (M-A251),, CD45RO (UCHL1), CD127 (HIL-7R–M21),, GATA3 (L50–823) and FOXP3 (236A/E7) antibodies as described ^{33 (link)}.