Anti epcam antibody clone g8

Single-cell suspensions were generated from peripheral lymph nodes (pLN, including inguinal, brachial, axillary, and submandibular, but not mLN), mLNs, spleen, and thymus by passing the tissues through 100 μm nylon filters (Merck Millipore). IELs from SI or large intestine (LI) were isolated as previously described.⁵⁵ In brief, SI and LI were harvested and washed in HBSS containing 2% FCS, followed by shaking in 25 mL of solution A media (10% FCS, 0.0154 g DL-dithiothreitol, and 1% 0.5 M EDTA in HBSS) at 240 rpm for 45 min. IELs from the SI were recovered from the supernatant of solution A media, and epithelial cells (ECs) were removed from the cell suspension by negative selection using anti-EpCAM antibody (clone G8.8, eBiosciences), followed by magnetic separation with BioMag beads (Qiagen). IELs from LI were recovered using 40/80% Percoll gradients (GE Healthcare) with centrifugation at 2,000 rpm for 25 min at room temperature. Cells were placed on ice until further analysis by flow cytometry. Where indicated, the SI was separated into two parts by cutting the junction between the jejunum and ileum as described previously before washing.^{56 (link)}