Live dead cell viability stain

Freshly isolated hHSC were stained with mAbs against CD19-APC-Cy7, CD146-FITC (BioLegend, London, UK), CD14-v500, CD45-FITC (BD Biosystems, Oxford, UK), CD68-FITC, Cytokeratin-FITC, CD3-PE-Cy7 (eBioscience, Hatfield, UK), CD56-ECD (Beckman Coulter, High Wycombe, UK), TRAIL-R1-PE, TRAIL-R2-PE, TRAIL-R3-PE and TRAIL-R4-PE (R&D Systems, Abingdon, UK) after FcR blocking reagent (Miltenyi Biotec, Surrey, UK) blocking. Appropriate isotype controls were used where necessary. NK cells were stained for CD56-ECD (Beckman Coulter, High Wycombe, UK), CD3-PE-Cy7 (eBioscience, Hatfield, UK), CD16-APC, TRAIL-PE. All cells were stained with Live/Dead® Cell viability stain (Invitrogen, California, USA). Data were acquired on BD^TM LSR II flow cytometer (Beckton Dickinson, New Jersey, USA) and analysed using FlowJo (Treestar Oregon, USA).

Polychromatic flow cytometry was used to analyze various B and Tfh cell subsets. Multiple Tfh subsets were determined using monoclonal antibodies including: CD3 Alexa Fluor 700, CD4 PE-Cy7, CD45RA BV605, CCR7 BV510, CXCR5 BV421, CCR6 FITC, CXCR3 APC, PD-1 PerCPCy5.5, ICOS PE. For B cell phenotyping, the following antibodies were used: CD19 FITC, CD20 BV421, CD10 PE, CD21 PE-Cy7, CD27 APC, CD24 BV650, CD38 PerCPCY5.5, CD3 APC-Cy7, FcRL4 PerCpCy5.5, FcRL5 PE, PD-1 BV421 (from Biolegend, eBiosciences or BD biosciences). All panels included staining with Live/Dead Cell viability stain (Invitrogen, California, USA) followed by staining with combinations of antibodies at 4 °C. Stained cells were washed with PBS and fixed in 1% paraformaldehyde till data acquisition on FacsAria. Data were analyzed using FlowJo (Treestar Oregon, USA).

Granulocytes were washed once with PBS and stained with Live/Dead cell viability stain (L3496570, Invitrogen, ThermoFisher Scientific) for 15 minutes at room temperature. Cells were again washed with 4 volumes of cold PBS (10010, Gibco) and tubes spun at 350g for 10 minutes at 4⁰C and then diluted to 20 million cells/mL in PBS. 10–15 million cells were stained with anti-human CD16 AlexaFluor® 647 (561724, BD Bioscience) and anti-human CDw125 PE (555902, BD Bioscience) for one hour at 4⁰C. Cells were washed with PBS (as previously described), diluted to 15 million cells/mL in PBS and sorted on a SH800S Cell Sorter (SONY Biotechnology) with 100 μm microfluidic sorting chip. Eosinophil (CDw125⁺,CD16^-) and neutrophil fractions (CDw125^-,CD16⁺) were collected. The purity of sorted cell fractions was assessed with flow cytometry on a FACS Fortessa instrument (BD Bioscience; S1 Fig) before being spun down and lysed in buffer (0.056 M sodium acetate, 1 M NaCl, 1% Triton X-100, pH 4.5). Lysates were kept at -80⁰C until the determination of EDN content with the ALPCO immunoassay.