Anti cd45 beads

Intracellular cytokine expression was measured as described previously.⁷ (link) Cells were stimulated with IL23 (10–20 ng/mL), IL6 (10–100 ng/mL), or phorbol 12-myristate 13-acetate (PMA) (50 ng/mL) and ionomycin (1 μmol/L) for 4–6 hours at 37°C with monensin (3 μmol/L) added for the last 2 hours. In human work, antibodies used to stain cell surface antigens were incubated with unstimulated cells for 25 minutes and then fixed in 2% paraformaldehyde pending analysis. For fluorescence-activated cell sorter purification of murine ILCs, CD45⁺ cells first were sorted immunomagnetically from unfractionated cLPMCs using anti-CD45 beads (Miltenyi) and LS columns (Miltenyi). CD45⁺ cells were stained with CD90, NKp46, and IL7R. Antibodies used in flow cytometry experiments are listed in Supplementary Table 1.

Single cell suspensions from BM (femur and tibia) and spleen were prepared. Splenic CD25⁺ cells were enriched with anti-CD25-PE and anti-PE magnetic beads using MACS® (Miltenyi Biotec, Bergisch Gladbach, Germany), stained for CD4 and CD62L and sorted on a FACS-ARIA II® (BD Biosciences, Heidelberg, Germany) as CD4⁺ CD25^– Tconv and CD4⁺ CD25^highCD62L⁺ Treg cells; purity was >98%.
For intestinal leukocyte isolation, small and large intestines were excised, Peyer’s patches removed, cut into 1–3 cm pieces, incubated twice for 15 min at 37 °C in HBSS/5mM EDTA/1mM DTT (Sigma-Aldrich, Munich, Germany) then vigorously shaken to isolate intraepithelial leukocytes (IEL). For lamina propria leukocytes (LPL), the fragments were transferred to DMEM with 0.13 U/ml Liberase^TM and 100 U/ml DNAse (Roche, Mannheim, Germany), mechanically dissociated, incubated at 37 °C for 30 min, further dissociated using GentleMACS® (Miltenyi Biotec), strained and then washed before Percoll centrifugation (75%/45%; IEL and small intestinal LPL) or magnetic separation using anti-CD45 beads (Miltenyi Biotec; colonic LPL).

Platelets were isolated as recently described in [23 (link)]. To remove residual red blood cells and leukocytes for pure platelet preparations, cells were incubated with anti-Ter-119 and anti-CD45 beads (Miltenyi Biotec, Bergisch Gladbach, Germany), respectively.

Splenic single-cell suspensions were prepared by mechanical disruption. The isolation of stromal cells from spleen has been described68 (link). Briefly, spleens were flushed with RPMI containing 3 mg ml⁻¹ Collagenase IV (Worthington), 40 μg ml⁻¹ DNAseI (Roche) and 2% (vol/vol) FCS), were cut into pieces and digested. Erythrocytes were lysed in 0.15 M NH₄Cl/10 mM KHCO₃/0.1 mM EDTA and haematopoietic CD45⁺ cells were depleted using anti-CD45 beads (Miltenyi Biotec). For TIL analysis, tumours were cut and digested with accutase (PAA), Collagenase IV (Worthington), Hyaluronidase (Sigma) and DNAseI (Roche) and red blood cells were lysed. Mononuclear cells were isolated using a Histopaque-1119 gradient.

Splenic B-cells were negatively selected via MACS with anti-CD43 beads (Miltenyi Biotec). Cells were cultured in RPMI 1650 media, 10% FBS, 0.00035% BME, 1% penicillin-streptomycin and stimulated with 10 ng/ml LPS and 20 ng/ml IL-4 for 72 hours. Cells were characterized by FACS (CD43- / B220+ / IgD+, S5 Fig). To generate bone marrow macrophages (BMMs), whole bone marrow was cultured in αMEM, 10% FBS, 1% penicillin-streptomycin, 50 ng/ml MCSF for 3 days. Cells were characterized by FACS (GR1- / F4/80+, S7 Fig). Proliferation was measured by standard MTT assay (Sigma-Aldrich). M2 polarized macrophages were generated by stimulating BMMs with 5 ng/ml IL-4 for 24 hours. Bone marrow stromal cells (BMSCs) were generated by plating whole bone marrow cells in ascorbic acid-free αMEM, 10% FBS, 1% penicillin-streptomycin for 7 days in 5% oxygen followed by negative selection via MACS with anti-CD45 beads (Miltenyi Biotec). Cells were characterized by FACS (CD45-, S6 Fig). Flow Cytometric Analysis was performed using FACSCALIBUR (BD Biosciences) and analyzed with FlowJo software (Tree Star). Antibodies used: APC-CD45 (BD Pharmingen), FITC-Gr1 (eBioscience), APC-F4/80 (BioLegend), FITC-CD43 (BD Pharmingen), APC-B220 (eBioscience), and PE-IgD (eBioscience).

For TEC analysis, single-cell suspensions were generated by digesting thymic lobes with collagenase Dispase (2.5 mg/ml; Roche) and DNase 1 (40 mg/ml; Roche). CD45⁻ cells were enriched by the depletion of CD45⁺ cells using anti-CD45 beads and LS columns (Miltenyi Biotec). The following Abs were used for TEC analysis: anti-CD45 clone 30-F11 (eBioscience), anti-EpCAM1 clone G8.8 (eBioscience), anti-Ly51 clone 6C3 (BioLegend), anti–MHC II clone M5/114.15.2 (eBioscience), anti-CD80 clone 16-10A1 (BioLegend), CD104 clone 346-11A (BioLegend), and anti–MHC I 28-14-8. Biotinylated UEA-1 (Vector laboratories) was detected using streptavidin PECy7 (eBioscience). Cells were analyzed using a LSR Fortessa (Becton Dickinson) with data analysis carried out using FlowJo v10 (Becton Dickinson). For cell sorting, TEC subsets were identified using the earlier Abs and isolated using a FACSAria Fusion 1 cell sorter (Becton Dickinson). The sorting strategy for the different TEC subsets was as follows: Cxcl12^DsRed+ cTEC, CD45⁻EpCAM1⁺UEA1⁻Ly51⁺CXCL12^DsRed+; CXCL12^DsRed− cTEC, CD45⁻EpCAM1⁺UEA1⁻Ly51⁺CXCL12^DsRed−; mTEC^lo, CD45⁻EpCAM1⁺UEA1⁺Ly51⁻CD80⁻MHC II⁻; mTEC^hi, CD45⁻EpCAM1⁺UEA⁺Ly51⁻CD80⁺MHC II⁺; CD104⁺ mTEC^lo, CD45⁻EpCAM1⁺UEA1⁺Ly51⁻CD80⁻MHC II⁻CD104⁺; and CD104⁻ mTEC^lo, CD45⁻EpCAM1⁺UEA1⁺Ly51⁻CD80⁻MHC II⁻CD104⁻.