Ly6c bv421

To generate a single cell suspension, colonic mucosa was isolated and digested for 30 min at 37 C in 100 ml DMEM with 2% heat-inactivated FBS, 0.2 mg/ml dispase II (Sigma, D4693), 2 mg/ml collagenase D (Roche, Indianapolis, IN, USA, #11088882001), and 0.2 mg/ml DNase I (Sigma, DN25) as previously described.^{59 (link)} For population analysis experiments, cells were fixed with 4% formaldehyde followed by permeabilization with 0.01% saponin. Cells were incubated with FcBlock (BD Biosciences, 553142, 1:100) for 15 min in FACS buffer (PBS+1% heat-inactivated FBS), followed by incubation for 30 min with the following fluorophore pre-conjugated antibodies: Ly6G FITC (Life Technologies, A25990, 1:100), F4/80-Alexafluor 488 (Life Technologies, MF48020, 1:100), CD11b-APC (Life Technologies, RM2805, 1:100), and Ly6C-BV421 (BD Biosciences, 562727, 1:100). Cells were also incubated with primary antibody against monoclonal ErbB4 (Abcam, ab32375, 1:40) for 30 min followed by anti-rabbit PE (Abcam, A10542, 1:100) for 30 min. Cells were analyzed on an LSR II flow cytometer (BD Biosciences).

Tumours were minced and digested in 3 mL RPMI with 2% Hepes, 2% FCS, 0.5 mg/mL Collagenase D and 0.1 mg/mL DNAse 1 at + 37 °C on a shaker (enzymes from Roche). After 45 min 300 μL 0.1 M EDTA was added for 5 min to stop the reaction. Single cell suspension was then obtained by using gentleMACS C tubes with a gentleMACS Dissociator and filtering of the suspension. Cells were blocked with BD’s FC-block (Cat# 553141) 30 min on ice, stained with directly conjugated antibodies from BD (30 min on ice) and a viability dye, recorded on a LSR Fortessa flow-cytometer (BD, BioSciences) and analysed with Flowjo v10 (FlowJo LLC). The used antibodies and dyes were: Fixable Viability Dye eFluor 780 (eBioscience, Cat# 65-0865-14), CD11b FITC (BioLegend, Cat# 101206), Ly6G PerCP-Cy5.5 (BD, Cat# 560602), F4/80 APC (eBiosicence, Cat# 17-4801-82), MHCII PE (BD, Cat# 557000), Ly6C-BV421 (BD, Cat# 562727).

Cell suspensions of spleen and peritoneal cells were stained with fluorescent antibodies (Abs) for 10 min at 4°C for flow cytometry analyses. Analysis of 200,000 events gated on viable cells was performed on a BD LSRFortessa Cell Analyzer (BD Biosciences, NJ, USA). Results were analyzed with BD FACSDiva Software 6.0 (BD Biosciences). Fluorescent Abs (TCRβ-Biot, SAV-BV605, CD4-PB, NK1.1-PerCPCy5.5, CD19-FITC, CD45-APC, CD45-BV711, CD11c-PECy7, Ly6C-BV421 and Ly6G-APC) were obtained from BD and CD8-APCvio770, CD11b-FITC and F4-80-PE were obtained from Milteyni.