Cxcr5 alexa fluor 647

To determine the percentage of different T cell subsets, at least 1 × 10⁶ PBMCs were suspended in isoflow sheath fluid (Beckman Coulter) and stained in a DuraClone IM T cell tube (Beckman Coulter, Miami, FL) according to the manufacturer's protocol. The T cell tube contained anti-CD45, CD3, CD8, CD4, CD45RA, and CCR7. Samples were measured by use of the Navios flow-cytometer (Beckman Coulter).
To determine the Tfh, at least 1 × 10⁶ PBMC were washed by Fascflow (BD Biosciences, New Jersey, US) and stained with CD3 BV510 (Biolegend, California, US), CD4 BV421 (Biolegend, California, US), CXCR5 Alexa Fluor 647 (BD Biosciences, New Jersey, US), and PD1 APC-Cy7 (Biolegend, California, US) for 30 min at room temperature in the dark.

CD4 cells were enriched using a CD4 T cell isolation kit, according to the manufacturer´s instructions (Miltenyi Biotec GmbH, Germany). The CD4 enriched cells were separated into two populations by flow cytometric sorting using anti-CD4-PacificBlue™, anti-CD45Ra-FITC and CXCR5-Alexa-Fluor 647 (all purchased from BD Bioscience). The resulting populations were routinely >98% pure.

Fluorochrome-conjugated monoclonal antibodies (mAbs) used in this study were CD45-PerCP and -Alexa Fluor 700, EpCam-FITC and -BV510, CD3-PerCP-Cy5.5, CD4-PE, -PE-Cy7, -BV786, and -AlexaFluor 700, CD8-APC-Cy7, CD45RA-BV605, HLA-DR-FITC, and -APC-Cy7, CD38-APC and -PE-Cy7, CD25-APC and -BB515, CD127-PE-CF594, CD103-PE, CXCR5-Alexa Fluor 647, CD16-BV421, and CD56-APC from BD Biosciences (San Jose, CA, USA); CD4⁺ 5RA-ECD (clone 2H4) from Beckman Coulter (Hialeah, FL, USA); CD127-eFluor450 and CD62L-APC-eFluor780 from eBioscience (San Diego, CA, USA); PD-1 (PE or APC, clone EH-12) and CD127-BV421 and -PE-Cy7 from BioLegend (San Diego, CA, USA); and IgA-PE and CD161-APC and -PE-Cy7 (Miltenyi Biotec, Germany). All antibodies were used according to the manufacturers’ directions.
For detection of surface markers, gut biopsy cells were stained with fluorochrome-conjugated antibodies for 15 min at room temperature, washed once with PBA [Dulbecco’s phosphate-buffered saline (DPBS) containing 0.5% BSA and 0.1% sodium azide], and resuspended in 0.5% paraformaldehyde (Electron Microscopy Sciences, PA, USA) in DPBS (PFA) for fixation, as previously described for human immunophenotyping (40 (link)). Intracellular staining for Ki-67 was performed as previously described (40 (link)), and intracellular staining for Bcl-6 was performed as previously described (31 (link)).