Liberase tl research grade

Stromal vascular fraction (SVF) cells were isolated by a previously described methodology^{20 (link)}. Briefly, small pieces of adipose tissue (1–2 g of SAT or MAT) (avoiding blood vessels) were added to tubes containing Hanks’ balanced salt solution supplemented with 4% BSA, 10 mM HEPES and Liberase™ TL Research Grade (Roche Diagnostics, Risch-Rotkreuz, Switzerland) and incubated in a water bath up to 60 min at 37 °C, with manual shaking each 10 min. After water bath incubation, digested samples were homogenized to single-cell suspensions, passed through a 100-μm cell strainer (BD Biosciences Pharmingen, San Diego, CA) and centrifuged at 280 × g for 10 min at 4 °C. Cells at the bottom, corresponding to the SVF were resuspended in Dulbecco’s PBS, supplemented with 2% FBS (Biowest, Nuaillé, France), 2 mM EDTA and 10 mM HEPES (all from Sigma-Aldrich) for flow cytometry studies and complete RPMI medium for cell sorting experiments.

Fragments of lungs (n = 5 mice) were collected, treated with 20 U/mL of DNAse I (GE Healthcare, Illinois, USA) and 0.13 mg/mL of liberase TL Research Grade (Roche – Sigma-Aldrich) and incubated in a ThermoMixer by 1000 rpm for 45 minutes at 37 °C. Subsequently, the reaction was stopped with RPMI 1640 medium supplemented with 10% fetal bovine serum and 1.5% of a solution containing 10000 U penicillin and 10 mg streptomycin/mL, and filtered through a 70 μm cell strainer. After that, the solution containing the cells were then centrifuged. Cell resuspension was adjusted to 1×10⁶ cells/well for flow cytometry analysis. Fragments of lungs were collected and added 1 mL of cytokines extraction solution [0.4 M NaCl, 0.05% Tween 20, 0.5% bovine serum albumin (BSA), 0.1 mM phenylmethanesulfonyl fluoride (PMSF), 0.1 mM benzethoniumchloride, 10 mM disodium ethylenediaminetetraacetic acid (EDTA) and 20 Kallikrein inhibitor (KI) aprotinin] to each 100 mg of tissue. Then, the Ultra-Turrax homogenizer-dispenser was used to homogenize solutions containing the organs. Subsequently, the samples were centrifuged at 10000 × G for 10 minutes at 4 °C. Cytokine production was evaluated in the supernatant using the Duoset ELISA kit (R&D Diagnostic) according to the manufacturer’s instructions. Two independent experiments were performed with n = 5 mice.

LRLNs and lungs were aseptically removed from euthanized mice and collected into 2ml tubes containing 1ml of incomplete media (ICM): RPMI-1640, 10 mM HEPES buffer, and 10 mM penicillin/streptomycin. These tissues were then mechanically homogenized and filtered through 70-µm cell strainers (Fisherbrand) to obtain single-cell suspensions. Lung tissue was additionally digested with 20 μg of Liberase TL research-grade (Roche) and 50 units of RNase-free DNase I (Promega) for 45 min at 37°C under 5% CO2, followed by 0.5M EDTA treatment for 5 mins. Cells were then suspended in freshly made ammonium-chloride-potassium (ACK) lysis buffer for 3 mins, then washed with ICM, and resuspended in complete media (CM): ICM plus 10% fetal bovine serum (FBS), 10 mM nonessential amino acids, and 10 mM sodium pyruvate. For flow cytometry analysis, single-cell suspensions were either stimulated overnight with heat-killed RB51 (HKRB51) followed by 4 hours of 5 ng/ml phorbol myristate acetate (PMA; SIGMA-ALDRICH), 500 ng/ml ionomycin (SIGMA-ALDRICH) and 10 μg/ml brefeldin A (SIGMA-ALDRICH) before antibody (Ab) staining or stained directly.