Red blood lysis buffer

Eight weeks after the 5/6Nx operation, the heart was exposed and perfused with 10 mL of PBS from the left ventricle. After removing the atria, the ventricles were digested in PBS containing 500 μg/mL of collagenase type II (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 200 μg/mL of CaCl₂ (Nacalai Tesque, Kyoto, Japan), 0.05% trypsin (Sigma-Aldrich), and 10% FBS at 37 °C for 10 min with agitation. Digested samples were further dissociated by passing them through a 23-gauge needle three times. After treating with Red Blood Lysis buffer (BioLegend, CA, USA), isolated cells were filtered through a 40 μm strainer.

Visceral adipose tissue (VAT) was collected from OB-ND, OB-Dys and LC during surgery. In particular, omental fat was collected from obese patients and perirenal fat from LC. Blood was withdrawn before general anesthetic injection. VAT was transferred to the laboratory in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with GlutaMAX (ThermoFisher – Scientific), bovine serum albumin (BSA), penicillin/streptomycin (P/S), and HEPES, where it was processed within 90 min from the collection. VAT was finely minced and digested with collagenase IV (Sigma-Aldrich) resuspended in PBS with a final concentration of 2 mg/mL for 40 min at 37°. The sample was then washed with DMEM high glucose (10% FBS, 1% P/S, 1% Glutamine) and treated with Red Blood Lysis buffer (Biolegend) to obtain the stromal vascular fraction (SVF). After washing, the pellet of SVF was filtered and counted. Whole blood (WB) was collected simultaneously with VAT and used for flow cytometry analysis. Peripheral blood mononuclear cells (PBMCs) were obtained using Lympholyte Cell Separation density gradient centrifugation media (Cedarlane). After centrifugation, the PBMC ring was collected and washed two times with PBS.

After sacrifice, spleen and lung samples were mashed through 70 µm strainer (22363548, Fischer scientific) to a new well. The strainer was washed with MAC buffer (2mM EDTA, 0,5% BSA, 1 x PBS). Isolated cells were incubated for 5 min RT with red blood lysis buffer (420301, Biolegend). The reaction was stopped with 1 x PBS. 1 x 10⁶ cells were spined down (5 min, 500 x G) and resuspend in 2% BSA, 1 x PBS, 2 µL TruStain FcX (101320, Biolegend). Total cellular fraction isolated from lungs were analyzed from the presence of lymphocytes. Specifically, the isolated cells were incubated with anti-CD8 and anti-CD19 antibodies according to manufacturer’s recommendations (Table S2). Isolated spleen cells were incubated with conjugated CD19/CD3 antibodies (Table S2) for 1 h at 4°C in dark. Blood was drawn with intracardiac punctures into anti-coagulated K2E tubes (BD Microtainer, 1307939). Whole blood was stained with conjugated anti-CD19 antibody for 1 h at 4°C in dark (Table S2). All samples were washed with cell staining buffer (BioLegend, 420201) and centrifugated for 5 min at 500 g. Cell pellet was resuspended in 500 µL of cell staining buffer. The presence of CD19-positive cells was analyzed using flow cytometry (BD LSRFortessa, BD Biosciences). The data was analyzed with Flowing Software 2.5.1 (Turku, Finland).