Ls column

Lung tissue was cut into small pieces (1 mm³) and treated overnight at 4 °C with Trypsin/EDTA (0.25%; Gibco), penicillin/streptomycin (100 U/mL; 100 µg/mL), 2 mg/mL Collagenase A (Roche, Basel, Switzerland) and 0.04 mg/mL DNase (Sigma-Aldrich). Next, the suspension was diluted in DMEM containing 0.04 mg/mL DNase and penicillin/streptomycin, homogenized and filtered over two layers of gause filter (Sefar Nitex, Heiden, Switzerland). After centrifugation (500× g for 10 min) the pellet was resuspended in lysis buffer (15.5 mM NH₄CL, 1 mM KHCO₃, 0.01mM EDTA, 0.04 mg/mL DNase) for 10 min at 4 °C. The remaining pellet was resuspended in Small Airway Epithelial Cell Growth (SAGM) medium (PromoCell, Heidelberg, Germany) supplemented with penicillin/streptomycin, and kept on ice until use within 1–4 hrs. Cell suspensions were seeded into organoid cultures directly or EpCAM⁺ cells were isolated by anti-CD45 (#130-045-801, Miltenyi Biotec, Auburn, AL, USA) and anti-CD31 (#130-091-935, Miltenyi Biotec) coupled-microbeads and passed through LS columns (Miltenyi Biotec) according to the manufacturer’s guidelines. The flow-through was incubated with anti-EpCAM (CD326) microbeads (#130-061-101, Miltenyi Biotec) for positive selection with LS columns. Cells were resuspended in SAGM and kept on ice until use within 1–2 h.

Lineage negative and CD31 positive BMCs were isolated using the first immunomagnetic Lineage Cell Depletion Kit (130-092-211; Miltenyi Biotec) followed by positive selection with CD31 MicroBead Kit (130-091-935; Miltenyi Biotec) using a magnetic cell separation device (QuadroMACS Separator;130-090-976; Miltenyi Biotec). Briefly, BMC suspensions were incubated for 10 min at 4 °C with Biotin-Antibody Cocktail, washed with MACS Buffer, and then incubated for 15 min with anti-Biotin MicroBeads. After washing, cells were separated on LS columns (130-042-401; Miltenyi Biotec) for positive and negative selection, respectively, according to the manufacturer’s instructions. After counting lineage negative cells, a second separating step with CD31 followed. Lineage negative cells were incubated with FcR Blocking Reagent and CD31 MicroBeads for 15 min at 4 °C. After washing, cells were separated on LS columns (130-042-401; Miltenyi Biotec) for positive and negative selection, respectively, according to the manufacturer’s instructions. Purity (Lineage and CD31 expression) of sorted fractions was checked by FACS-analysis with BV421-conjugated CD31 (303124; Biolegend; clone WM59) antibody and FITC-conjugated Lineage cocktail 4 (562722; BD; clones RPA-2.10, HIT3a, RPA-T4, M-T701, HIT8a, B159, GA-R2 (HIR2)).

Peripheral blood mononuclear cells (PBMCs) were obtained from blood by Ficoll-Paque density gradient centrifugation according to manufacturer’s protocol. CD14⁺ monocytes were isolated from PBMC using anti-CD14 MicroBeads, LS+ columns and MACS separators (Miltenyi Biotec). Monocyte-derived DCs (referred as DCs) were differentiated from CD14⁺ monocytes by culture with rhIL-4 and rhGM-CSF (at 500 and 1,000 U/ml, respectively, R&D Systems) during 7 days. Sequentially after the CD14⁺ isolation, CD4⁺ T cells and NK cells were isolated using anti-CD4 and NK cells isolation MicroBeads, respectively, LS+ columns and MACS separators (Miltenyi Biotec). CD4⁺ T cells and NK cells were frozen in 90% fetal calf serum (FCS; PAA) and 10% DMSO (Sigma). The percentage of NK cells and CD4⁺ T cells after isolation was 95% by flow cytometry. We have previously determined that, in our settings, NK cell activity is not affected by freezing (12 (link), 13 (link)). After thawing, NK cells were cultured overnight in medium prior to the priming. In all experiments, cells were cultured in DMEM supplemented with 10% FCS (PAA), 10 mM HEPES, 1× non-essential amino acids, 100 μg/ml streptomycin, 100 U/ml penicillin, 1 mM sodium pyruvate, 2 mM l-glutamine, 1× vitamins, and 50 μM 2-mercaptoethanol (Invitrogen).

After collagenase (Sigma, USA) digestion of WAT and BAT harvested from 4 mice, large adipocytes were separated by low speed centrifugation. Next, magnetic separation was performed according to the manufacturer’s instructions using an autoMACS pro Separator (Miltenyi Biotec GmbH, Germany). The cell pellet was re-suspended in 900 μl MACS sorting buffer and incubated with 100 μl Cd11b MicroBeads (MiltenyiBiotec GmbH, Germany) per 10⁷ total cells at 4 °C for 15 min. After centrifugation, the cell pellet was re-suspended and cell suspension was applied on LS columns (MiltenyiBiotec GmbH, Germany). The magnetically labeled Cd11b+ macrophage cell fraction was collected from LS columns. The flow through fraction was incubated with Cd31 MicroBeads (MiltenyiBiotec GmbH, Germany) and applied on LS columns to collect Cd31+ endothelial cells. The last flow through fraction was obtained as adipocytes.