Nylon mesh

AT components were collected from lipoaspirates using a modified Rodbell’s protocol as described in the Appendix II of Sanna et al. [63 (link)]: AT was directly digested in a collagenase type 2 solution (1 mg/mL, Merck, Darmstadt, Germany) in DMEM F12 (ThermoFisher Scientific, Waltham, Massachusetts, USA) for 30 min at 37 °C through gentle stirring. Then, tissue was mechanically disrupted using a pipette and the digested tissue was filtered through a nylon mesh of 100 µm pore size (Corning, NY, USA). After low speed (100× g) centrifugation, the upper phase was collected and stored as adipocytes (AD). The residual volume was then centrifuged at 300× g, and the pellet was collected as a stromal vascular fraction (SVF). SVF was then subjected to a red blood cell lysis using a hypertonic buffer. After the last centrifugation, the recovered cells were then counted.

Subcutaneous ingWAT was removed, weighed, and placed in a digestion buffer. Cells went through the digestion of the tissue by collagenase [28 (link)] with some modifications. Briefly, the fat pad was digested in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with HEPES (20 mM), sodium pyruvate (2 mM), bovine serum albumin (BSA, 1%), and collagenase type II (1 mg/mL), pH 7.4 at 37 °C in an orbital bath shaker for about 45 of 60 min. After elimination by filtration through a nylon mesh (Corning, NY, USA) of undigested fragments, the filtrate was centrifuged (400× g, 1 min), and then divided into two fractions: 1. The floating adipocyte layer, and 2. The stromal-vascular fraction (SVF) cells (all remaining filtrate), which was subjected to centrifugation (1500× g for 10 min). Cells were pooled from two mice. One pooled cell was counted as one sample. Isolated mature adipocytes were washed three times in fresh buffer without collagenase. After washing and brief spin, the medium was thoroughly aspirated, and adipocytes were harvested. Aliquots of isolated adipocytes suspensions were placed in a microscope slide and 6 fields were photographed under an optical microscope (×100 magnification) coupled to a microscope camera (AxioCam ERc5s; Zeiss, Oberkochen, Germany), and mean adipocyte volume (4/3 × π × r3) was determined by measuring 100 cells using AxioVision LE64 software.