Il 11

Purified PreGM, GMP and PreMegE cells were cultured at 37°C in 5% CO₂ and atmospheric O₂ for 6–18 hours in pro-myeloid medium (IMDM supplemented with 10% FBS for Mouse Myeloid Colony-Forming Cells, 10% Protein-free hybridoma medium (PFHM-II, Gibco), 180μg/ml Transferrin (Roche Diagnostics), 0.45mM MTG, 50ng/ml ascorbic acid (Sigma-Aldrich), 2mM L-glutamine, 1x Penicillin-Streptomycin, 4U/ml erythropoietin, 5ng/ml IL11 (R&D Systems), 10ng/ml IL6 (R&D Systems), 10ng/ml M-CSF (R&D Systems) and medium conditioned by cell lines producing IL3, GMCSF, TPO and SCF (1% final concentration)). Purified LSK48F- MPPs were cultured for 12–24 hours in Alpha MEM (Lonza) supplemented with 10% FBS, 0.45mM MTG, 2mM L-glutamine, 1x Penicillin-Streptomycin, 1U/ml erythropoietin, and medium conditioned by cell lines producing IL3, TPO and SCF (1% final concentration)) (adapted from [70 (link)]). Cells were then harvested and stained with either LSK HSPC markers or myeloid progenitor markers as described in S2 Table.
For longer-term culture (7–11 days), PreGM and GMP cells were cultured in pro-myeloid medium as described above before being harvested and stained with GM and mast lineage markers (CD11B, GR1, F4/80, C-KIT and FCΕR1Α as detailed in S2 Table).
Sorted MkP cells were cultured for 4 days in pro-myeloid medium (see above).

Cryopreserved PBMCs were thawed and stained with viability dye (eFluor 780; eBioscience). Antibodies against CD4 (FITC or V500), CD25 (APC), CD45RA (Alexa Fluor 700)(BD Biosciences), CD127 (PE-eFluor 610), FOXP3 (PE), TIGIT (PerCP-eFluor710) (eBioscience), Helios (Pacific Blue; Biolegend). Purified anti-FCRL3 antibody was provided by Nagata (37 (link)), and was detected with F(ab′)2 anti-mouse IgG (PE-Cy7; eBioscience). Flow cytometry analysis was performed on an LSR Fortessa analyzer (BD Biosciences), and sorting throughout this study was performed on a FACS Fusion cell sorter (BD Biosciences). Recombinant human IL-6, IL-27, CLC, IL-11 (R&D systems), and LIF (Peprotech) were added to suppression assays where indicated at the time of activation.

Enzyme-linked immunosorbent assay (ELISA) was performed to detect the presence of cytokines IL-8 and IL-11. EA.hy926 cells (1.5 × 10⁵ cells/well) were plated in DMEM (2 mL) containing 10% FBS in 6-well plates. BEAS-2B cells (2 × 10⁵ cells/well) were plated in MEM (2 mL) containing 10% FBS in 6-well plates. The medium was then removed, and silver NPs suspended in the growth medium and silver NPs suspended in 2% FBS-containing growth medium were added to each well containing EA.hy926 and BEAS-2B cells, respectively, to obtain a final volume of 1 mL per well. ELISA was performed using Human Cytokine IL-8 (BD Biosciences, San Diego, CA, USA) and IL-11 (R&D Systems, Minneapolis, MN, USA) Assay Kits. These kits use biotinylated anti-IL-8 or anti-IL-11 antibodies and streptavidin conjugated to horseradish–peroxidase. The OD was measured at 450 nm.