Fortessa analyser

Tumors were mechanically dissociated and strained through a 40 µm nylon mesh before digestion into single cells with collagenase type II (0.5 mg mL⁻¹), collagenase type IV (0.5 mg mL⁻¹), hyaluronidase (10 U/mL) and DNase I (0.01 mg mL⁻¹) for 2 h at 37 °C. The dissociated cells were collected, lysed by RBC lysis buffer. Phenotypic analyses were carried out on isolated macrophages for distinct M1/M2 populations as indicated. Cells were stained in ice-cold PBS containing FCS (2%) and EDTA (2 mM) using appropriate antibody-fluorophore conjugates. Multiparameter analysis was performed in a Fortessa analyser (BD Biosciences) and analysed with FlowJo software (Tree Star, Ashland, USA). The following antibodies were purchased from Biolegend (San Diego, USA): anti-CD45 (clone 30-F11), anti-GR-1 (clone RB6-8C5), anti-F480 (clone BM8) anti-CD206 (clone C068C2) and anti-CD86 (clone GL-1) while anti-CD11c (clone N418) and Ly6G (clone Ia8) were purchased from ebioscience (ThermoFisher Scientific, Waltham, MA, USA).

MAD2L2 WT, MAD2L2 R124A, and MAD2L2 3xMut(K44A/R124A/A135D) vectors were introduced by retroviral infection and CH12 cells were selected with blasticidin (10 μg ml⁻¹). The aforementioned TRIP13-shRNA and control vectors were introduced by lentiviral infections and CH12 cells were selected with puromycin (3 μg ml⁻¹). CH12 cells were subsequently plated at 50,000 cells/ml in complete RPMI supplemented with anti-CD40 antibody (1 μg ml⁻¹, Miltenyi), IL-4 (20 ng ml⁻¹, Miltenyi) and TGF-β (1 ng ml⁻¹, R&D Biotech) to induce IgM to IgA switching. After 3–4 days, cells were assayed for class-switching by flow cytometry using an IgA-PE antibody (eBiosciences) and a Fortessa analyser (BD Biosciences). Viable cells were counted using a Casy cell counter (Roche). CSR and proliferation assays were done in at least three independent experiments.

Cells were washed with FACS buffer (PBS buffer supplemented with 1% FCS and 1 mM EDTA) by centrifugation and stained with the respective antibodies. Reactions were incubated for 30 min at 4 °C. Following two washes with FACS buffer, samples were resuspended in 200 μl of FACS buffer and data was acquired using a Fortessa analyser (BD Bioscience). All data were processed with FlowJo (v9.9, TreeStar). Antibodies used in this study: anti-human CD4, APC (BioLegend; Clone: OKT4, Catalog No: 317416, Dilution: 1:100), anti-human CD45RA, Brilliant Violet 785 (BioLegend; Clone: HI100, Catalog No: 304140, Dilution: 1:100), anti-human CD45RO, PE-Cyanine7 (BioLegend; Clone: UCHL1, Catalog No: 304229, Dilution: 1:100), anti-human CD197 (CCR7), (BD Bioscience; Clone: 150503, Catalog No: 561271, Dilution: 1:100), anti-human IFN gamma, PE-Cyanine7 (eBioscience; Clone: 4s.B3, Catalog No: 25-7319-82, Dilution: 1:50), anti-human IL-9, PE (BD bioscience; Clone: MH9A3, Catalog No: 560814, Dilution: 1:50).

CH12 cells were plated at 50,000 cells per ml in complete RPMI supplemented with anti-CD40 antibody (1 μg/ml, Miltenyi), IL-4 (20 ng/ml, Miltenyi), and TGF-β (1 ng/ml, R&D Biotech) to induce IgM to IgA switching. After 3 days, cells were assayed for class switching by flow cytometry using IgA-PE (eBiosciences 12-4204-82, clone mA-6E1, 1:200) and IgM-PE-Cy7 (BD Biosciences 552867, clone R6–60.2, 1:200 dilution) antibodies, and a Fortessa analyser (BD Biosciences). Data were analyzed by FlowJo v10.4.2 software. The gating strategy is provided in Supplementary Fig. 8.

Harvested cells were washed in ice-cold PBS, fixed in 70% ethanol (2 h at 4 °C) and washed in PBS. Cells were stained with propidium iodide (PI) solution (25 μg/ml PI, 20 μg/ml RNase A/T1, PBS) for 30 min at 37 °C, processed on a Becton-Dickinson Fortessa Analyser and analysed using FlowJo software.

For cell sorting: 30 × 10E6 cells were counted and resuspended in 300 μl PBS + 3% FBS in the presence of FcR blocking reagent. Cells were incubated for 10 min and 15 μl of the human anti-CD19 antibody conjugated with BV510 (Becton Dickinson, 562947) and 15 μl of human anti-cd11b (Mac1) antibody conjugated with PE-Cy7 (eBioscience, 25-0118-41) were added. Cells were incubated for 30 min in the dark, washed with PBS and resuspended in 2 ml of PBS + 3% FBS. Topro-3 was added as a viability marker. Cells were sorted in a BD FACS Aria instrument at the Flow Cytometry Unit of the Centre for Genomic Regulation.
For flow cytometry analysis: 1 × 10E6 cells were counted and resuspended in 100 μl PBS + 3% FBS in the presence of FcR blocking reagent. Cells were incubated for 10 min and 5 μl of each of the corresponding antibodies were added. For the CD19 knockout experiment, we used the antibody anti-CD19 conjugated with APC-Cy7 (Becton Dickinson, 557791). Cells were incubated for 30 min in the dark, washed with PBS and resuspended in 500 ul of PBS + 3% FBS. Topro-3 was added as a viability marker. Cells were measured in a BD Fortessa analyser. For the Stain Index calculation we used the formula: (mean positive—mean background) / (2 * SD background), as previously described [63 (link)].
Cell cytometry data is available in FlowRespository database (https://flowrepository.org) [64 (link)].