Gentlemacs c tube

For human T cells, PBMCs were isolated by centrifugation of blood over a Ficoll (Biotest, Dreieich, Germany) layer. Overall, 10 ml of Ficoll was loaded under 20 ml blood diluted with 20 ml phosphate-buffered saline (PBS) and centrifuged at 400 × g for 20 min at room temperature. PBMCs were harvested and washed once with PBS. Total CD4⁺ T cells, naive CD4⁺ T cells, CD25^- CD4⁺ T cells and CD25⁺ CD4⁺ T cells were purified from human PBMCs by magnetic cell separation (MACS) using the Human CD4⁺ T Cell or CD4⁺ CD25⁺ Regulatory T Cell Isolation Kit (Miltenyi Biotec).
For murine T cells, spleens were homogenized into a single-cell suspension using gentleMACS™ C Tubes and a gentleMACS™ Dissociator (Miltenyi Biotec). Cell populations were purified from splenocytes either by MACS or by fluorescence-activated cell sorting (FACS) on a MoFlo (Beckman Coulter, Brea, CA). Total CD4⁺ T cells were purified using the Mouse CD4⁺ T Cell Isolation Kit (Miltenyi Biotec). CD25⁺ CD4⁺ and CD25⁻ CD4⁺ T cells were purified using the Mouse CD4⁺ CD25⁺ Regulatory T Cell Isolation Kit (Miltenyi Biotec). The purity of the cell populations was evaluated by flow cytometry.

Mice were euthanized and perfused with 10 mL saline solution to remove
peripheral blood and ensure that leukocyte populations found are those residing
in the adipose tissue. Perigonadal VAT was isolated and washed with ice-cold
PBS. VAT was then minced to 2–3 mm pieces, added 4 mL of enzymatic
digestion mix and transferred to gentleMACS C-tubes (130-096-334; Miltenyi
Biotec, Bergisch Gladbach, Germany). Tissue was then dissociated using the
gentleMACS Octo Dissociator (130-095-937; Miltenyi Biotec, Bergisch Gladbach,
Germany), program name: mr_adipose_01, ran 3 times. Suspensions were
subsequently filtered with a 100 μm cell strainer, washed with ice-cold
PBS and stained for cell-sorting. Adipose enzymatic digestion mix contained 1
mg/mL bovine-serum albumin, 0.77 mg/mL Liberase (0541151001; Roche,
Indianapolis, USA), 15.8 mU Hyaluronidase (H3506; Sigma-Aldrich, St. Louis,
USA), 25 mU DNAse1 (DN25; Sigma-Aldrich, St. Louis, USA) and 1.5 μM
Ca²⁺ in Hanks’ Balanced Salt solution.

Three tail samples per time point (0, 1, 3, 7, 14, 21, and 28 DPA) were cut to 3–5-mm pieces in length and each piece was cut into 1/8ths. Tail pieces were added to gentleMACS™ C tubes (Miltenyi Biotec, PN: 130-093-237) containing 2.5 mL Dulbecco’s modified Eagle media (DMEM), 100 µL proprietary Enzyme D, 50 µL Enzyme R and 12.5 µL Enzyme A from Multi Tissue Dissociation Kit 1 (Miltenyi Biotec, PN: 130-110-201). Tubes were inverted and placed onto gentleMACS™ OctoDissociator with sleeve (Miltenyi Biotec, PN: 130-096-427) and a fibroblast dissociation protocol was run for 1 h. Enzymatic activity was inactivated with DMEM supplemented with 10% fetal bovine serum (FBS). Cells were gently resuspended via pipetting and run through MACS® 70 µm SmartStrainer (Miltenyi Biotec, PN: 130-098-462), followed by filtration via Scienceware FlowMi® 40 µM Cell Strainers for p1000 pipettes (Sigma Aldrich, PN: H13680-0040). Cells were pelleted and washed with DMEM with 10% FBS. Cells were resuspended in HBSS with 0.04% bovine serum albumin (BSA) prior to library preparation.

Lung perfusion was performed by injecting 10 ml of cold PBS through the right ventricle. Harvested lungs were collected in a solution containing Collagenase D (0.15 U/ml, Roche Life Sciences) and DNase (40U/ml, Roche Life Science) dissolved in a Hank’s Balanced Salt Solution (HBSS) supplemented with calcium and magnesium (Thermo Fisher). Lungs were subsequently placed in Gentle MACs C tubes and processed using the GentleMACS Dissociator (Miltenyi Bio-tec) for 30 minutes at 37°C. The cell suspension was filtered through a 40mm strainer and lysed using red blood cell lysis solution (RBC Lysis Buffer, Biolegend). Cells were washed twice with PBS, counted and used for assays. Spleen and mediastinal lymph nodes were collected in PBS and passed through a 40mm cell strainer. After lysis, cells were reconstituted in PBS for further use.