Cd3e percp cy5.5 clone 145 2c11

Fresh total cells from spleens of WT mice 8 weeks after infection with Sjaponicum were pre‐sorted by CD4⁺ T cell negative‐isolation kit (Miltenyi Biotec), and then stained with CD3e‐Percp‐cy5.5 (clone 145‐2C11, eBioscience), CD4‐FITC (clone GK1.5, eBioscience), PD‐1‐PE (clone J42, eBioscience) and CXCR5‐APC (clone 2G8, BD Pharmingen) antibodies. CXCR5^highPD‐1^high Tfh cells were FACS‐purified by using a FACSAria cell sorter (BD Biosciences) to investigate the effect of Tfh cells on the cellularity of granulomas in the liver. FACS‐sorted Tfh cells were resuspended in PBS and injected intraperitoneally (ip) into the ICOSL^−/− mice 5 weeks after Sjaponicum infection (3 × 10⁶ cells/mouse). A group of mice that did not receive T cells but PBS was used as an additional control (mock transfer). Mice were sacrificed 3 weeks after transfer.

Single‐cell suspensions from spleen, mesenteric lymph nodes and liver were isolated in phosphate‐buffered saline (PBS) containing 1% foetal bovine serum as described previously.12 For CXCL12⁺ Tfh cell analysis, cells were stimulated for 6 hours in culture medium containing phorbol myristate acetate (PMA, 25 ng/mL; Sigma‐Aldrich), ionomycin (1 μg/mL; Sigma‐Aldrich) and monensin (Golgi Stop; 1 μg/mL; BD Biosciences). Cells then were incubated for 30 minutes at 4°C with the following monoclonal antibodies: CD3e‐Percp‐cy5.5 (clone 145‐2C11, eBioscience), CD4‐PE‐Cy7 (clone GK1.5, eBioscience), PD‐1‐PE (clone J42, eBioscience) and CXCR5‐FITC (clone 2G8, BD Pharmingen). After staining of surface markers, the cells were permeabilized with cold Fix/Perm Buffer, and incubated with CXCL12‐APC (clone 79018, R&D Systems) after blockade with antimouse CD16/32 (clone 93, eBioscience). For eosinophil surface marker analysis, cells were incubated with Siglec‐F‐PE (clone E50‐2440, BD Pharmingen), CXCR4‐FITC (clone 2B11/CXCR4, BD Pharmingen) or isotype (BD Pharmingen) antibodies. Cells acquisition was performed using a FACSVerse cytometer (Lasers: 488 and 633; Mirrors: 507 LP, 560 LP, 665 LP, 752 LP, 660/10, and 752 LP; Filters: 488/15, 527/32, 568/42, 700/54, 783/56, 660/10 and 783/56, BD Biosciences). Data were analysed with FlowJo (Tree Star, version 10.0.7).