Anti cd103 clone ber act8

Cryopreserved single-cell suspensions were thawed at 37°C, aliquots of IEL/LPL fraction pooled (when applicable), and rested for 8 hours in RPMI/10% FBS. Single-cell suspensions were incubated with Fc-receptor block for 10 minutes at 4°C (TruStain FcX, BioLegend) followed by hashing antibody (0.5μg per 2 million cells) for 30 minutes at 4°C. Following multiple washes, samples were stained with anti-CD3 (clone OKT3, BioLegend) FITC, anti-CD4 (clone SK3, BD-Horizon) BUV737, anti-CD8α (clone RPA-T8, BioLegend) BV785, anti-CD45 (clone HI30, BioLegend) BV711, and anti-TCRγδ (clone 5A6.E9, Thermo Fisher) PE. Gluten challenge samples were stained with anti-CD38 (clone HIT2, BioLegend) APC, anti-CD103 (clone Ber-ACT8, BioLegend) BV711, and anti-Integrin β7 (clone FIB504, BioLegend) PE/Dazzle^™ 594. Samples were incubated with pre-titrated TotalSeq-C CITE-seq antibody cocktail (BioLegend, PN900000115) for 30 minutes at 4°C. LIVE/DEAD Fixable Red (ThermoFisher) and DAPI were used to gate viable cells. T cells were sorted into RPMI by FACS AriaII or Propel Labs Bigfoot Cell Sorter by gating viable CD3⁺ intestinal cells and CD3⁺CD4⁻CD38⁺CD103⁺ cells from gluten challenge samples. FACS analysis performed using FlowJo (v.10.7.1) software.