Cytospin 3 cytocentrifuge

For cytokine sensitivity assays, hematopoietic cells (Table S2) were plated at 2×10⁴ in methylcellulose (MethoCult® H4531, Stem Cell Technologies) with escalating concentration of GM-CSF (0.01, 0.1, 1 and 10 ng/mL) (R&D) or 1.5×10⁴ in methylcellulose containing agar leukocyte conditioned medium (Agar-LCM), formulated to support the growth of granulocyte and macrophage progenitors. The hypersensitivity to GM-CSF was observed in a dose-response curve, represented as percentage of maximal colony formation and calculated by dividing the number of colonies at each GM-CSF concentration by the average number of colonies at GM-CSF 10 ng/mL (Table S2). For decoy and over-expression miR-223 assays, the treated hematopoietic cells were plated at 2×10⁴ with 0.01 ng/mL GM-CSF (Table S2). After 14 days at 37 °C in humidified 5% CO₂, myeloid colonies were counted. For clonogenic myeloid progenitor assays, MethoCult® H4435 Enrich (Stem Cell Technologies) were used. Fourteen days after plating, myeloid and erythroid colonies were counted based on standard guidelines previously described (Nissen-Druey et al., 2005 (link)). Cell morphology was assessed using a Cytospin 3 cytocentrifuge (Thermo Fisher Scientific) and staining with Giemsa (Accustain, Sigma-Aldrich).

BALF cells were spun onto poly-L-Lysine slides with a Cytospin 3 cytocentrifuge (Thermo Scientific), fixed with 4% PFA for 10 min at room temperature, washed thrice with PBS and blocked / permeabilized with blocking solution (3% BSA with 0.1% Triton-X 100 in PBS) for 1 h at room temperature. Cells were incubated with 5 µg/ml of anti-γH2AX (Cell signaling) and 5 µg/ml of anti-F4/80 (Biolegend) in blocking solution for 1 h at room temperature, washed and stained with 10 µg/ml of Alexafluor dyes conjugated secondary antibodies (Molecular probes) for 45 min at room temperature, followed by staining with DAPI for 15 min.