Epcam clone g8.8

As described previously35 , subcutaneous tumours with KP1.9 cells were harvested from C57BL/6 flanks 3 weeks after implantation, minced, and shaken at 600 r.p.m. with 0.2 mg ml⁻¹ collagenase type I (Worthington Biochemical Corporation) in RPMI-1640 for 30 min at 37 °C. Digested samples were filtered (70 μm BD Falcon strainer); washed in PBS with 0.5% BSA and 2 mM EDTA; incubated with Fc-block (TruStain fcX anti-mouse CD16/32; clone 93; Biolegend) for 15 min at 4 °C; and labelled with antibodies as indicated for 45 min at 4 °C. Flow cytometry (LSRII, BD Biosciences) labelled tumour cells (CD45⁻ EpCAM⁺), TAM (CD45⁺ CD11b⁺ Ly6C^- Lin^- CD11c⁺ F4/80⁺), lymphocyte-like cells (CD45⁺ CD11b^- Lin⁺), along with CD45- EpCAM- host-cell populations. Antibodies included EpCAM (clone G8.8; eBioscience); CD45 (clone 30-F11; Biolegend), F4/80 (clone BM8; Biolegend), CD11c (clone N418; Biolegend), Ly6C (clone HK1.4; Biolegend); and CD11b (clone M1/70; BD Biosciences). The lineage (Lin) antibody mix contained anti-CD90.2 (clone 53–2.1), anti-B220 (clone RA3-6B2), anti-NK1.1 (clone PK136), anti-CD49b (clone DX5), anti-Ter119 (cloneTER-119) and anti-Ly6G (clone 1A8) (all BD Biosciences). 7-aminoactinomycin D (7-AAD, Sigma Aldrich) excluded dead cells. VT680 fluorescence was directly assessed using the LSRII flow cytometer, FlowJo v.8.8.7 (Tree Star, Inc.) and MATLAB.

Preparation of thymic epithelial cells was performed as previously described [76 (link)]. Thymic tissue were cut into small pieces by forceps and placed into 15 mL tube containing 2mL of RPMI-1640 (Sigma) + 1% FCS. After pipetting and settling for 2 min, the supernatant were discarded. This was repeated several times. RPMI-1640 + 1% FCS containing 0.5 U/mL Liberase TM (Sigma-Aldrich) and 0.02% (w/v) DNaseⅠ (Roche) were added to remaining thymic fragments and incubated at 37°C for 12 min. After settling for 2 min, the supernatant were collected into 15 mL tube and suspended with PBS (-) + 1% FCS + 5 mM EDTA. This step was repeated twice. After washing cells, they were passed through mesh. Single cell suspension was stained with CD45 (clone 30-F11, eBioscience), EpCAM (clone G8.8, eBioscience), Ly51 (clone 6C3, BD Pharmingen) and UEA-1 (Vector Laboratories) and sorted by using FACSAriaⅢ (Becton Dickinson).