Adipose Tissue Secretome Isolation

Adipose tissue samples (n = 78: 26 SAT, 26 VAT and 26 BAT) were processed as previously described with some modifications to obtain tissue secretomes [9 (link),66 (link)]. In brief, whole adipose tissue explants were processed to remove any contaminants by doing an intensive wash in PBS that was repeated several times. The tissue pieces were transferred to a tube containing 25 mL of PBS and centrifuged for 5 min, at 1800 rpm, at room temperature to eliminate red blood cells and debris. 1 g pieces of each tissue type (VAT, SAT, and BAT) from independent animals were incubated at 37 °C and 5% CO₂ in 5 mL/tissue piece/well with serum-free DMEM medium without phenol red (Sigma-Aldrich, Burlington, MA, USA) supplemented with 1% (v/v) penicillin-streptomicin. The medium was changed after 2 and 24 h. After the last wash (time point 24 h), all dishes received fresh DMEM medium (3 mL/1 g tissue) and were left in culture for an additional 48 h to allow the secretion of vesicles. Then, the media were collected, centrifuged during 5 min at 1800 rpms, and stored at −80 °C for further analysis.