Anti epcam pe

Prior to FACS staining, tumor spheroids were dissociated into single cells by trypsination for 10 min at 37 °C, followed by vortexing for 10 s. To analyze cell cycle distribution, tumor single cells were fixed in 70 % ice-cold ethanol, washed with PBS + 2 % FCS, and treated with 100 μL RNAse (1 mg/mL) at 37 °C for 20 min. Cells were stained with 10 μL propidium iodide (1 mg/mL) for 30 min. FACS analysis was performed on a FACS Canto II instrument using Diva Software (BD Biosciences). Proliferation was analyzed by FACS after staining tumor single cells with anti-Ki67 PEVio770, anti-CD45 FITC, and anti-EpCAM PE antibodies (all Miltenyi Biotech).

Cell suspensions were incubated for 15 min at 4°C with Fc Block (BD Biosciences) and then stained for 25 min at 4°C with the following fluorochrome-conjugated antibodies: anti-CD11c-APC-Cy7 (clone: HL3, BD Biosciences) or anti-CD11c-PE (clone REA754, Miltenyi Biotec), anti-MHC-II-VioBlue (clone: M5/114.15.2, Miltenyi Biotec), anti-CD11b-PerCP-Vio700 (clone: REA592, Miltenyi Biotec), anti-EpCAM-PE (clone: caa7-9G8, Miltenyi Biotec) or anti-EpCAM-PE-Vio770 (clone caa7-9G8, Miltenyi Biotec), anti-XCR1-APC-Vio700 (clone REA707, Miltenyi Biotec), anti-PD-L2-PE (clone MIH37, Miltenyi Biotec), CD86-APC (clone PO3.3, Miltenyi Biotec). Dead cells were excluded using Zombie Aqua Fixable Viability Kit (Biolegend). For the analysis of Fc receptor expression, cells were permeabilized using an intracellular fixation & permeabilization kit (eBioscience) and incubated for 25 min at 4°C with anti-CD16(FcγRIII)/CD32(FcγRII)-PE-Vio770 (instead of Fc Block, clone: 93, Miltenyi Biotec), anti-FcϵRIα-APC (clone: MAR-1, Miltenyi Biotec), anti-CD23-APC (clone: B3B4, Miltenyi Biotec), anti-CD64-PE-Vio770 (clone: REA286, Miltenyi Biotec). Cells were acquired on a MACSquant 10 or a MACSquant 16 flow cytometer (Miltenyi Biotec) and data were analyzed using FlowJo software using the gating strategies described in Figures S2 and S3 (10 (link)).