Isolation and Characterization of Adipocytes

Tissue samples of EAT and SAT were obtained in the course of CABG in the amount of 0.2–1 g as described before [21 (link)]. Briefly, the samples were placed in M199 medium preheated to 37 °C in advance and delivered to the laboratory within 15 min. Adipocytes were isolated enzymatically in sterile conditions (laminar box BAVp-01- “Laminar-s” −1.5, ZAO “Laminar systems”, Miass, Russia) [22 (link)]. Adipose tissue was minced and incubated during 35–40 min at 37 °C and constant mild shaking (10 rpm) in 5 mL of collagenase type I solution (PanEco, Moscow, Russia) 1 mg/mL in Krebs–Ringer buffer (2 mM D-glucose, 135 mM NaCl, 2.2 mM CaCl₂·2H₂O, 1.25 mM MgSO₄·7H₂O, 0.45 mM KH₂PO₄, 2.17 mM Na₂HPO₄, 25 mM HEPES, 3.5% BSA, 0.2 mM adenosine). Krebs–Ringer buffer (37 °C) was added to the digested tissue to neutralize collagenase in 1:1 ratio. The suspension of cells was filtered through the nylon mesh (Falcon™Cell strainer, Glendale, AZ, USA, 100 μm) and rinsed three times with warm Krebs–Ringer buffer. The numbers and size of the obtained adipocytes were calculated in a Goryaev cell chamber by light microscopy (Axio Observer.Z1, Carl Zeiss Surgical GmbH, Oberkochen, Germany). The representative images of EAT adipocytes are displayed in Figure 1. Cells were stained with Hoechst 33,342 (5 μg/mL, stains nucleus of viable cells) and propidium iodide (10 μg/mL, Sigma-Aldrich, St. Louis, MO, USA, stains nucleus of dead cells) to distinguish viable cells from dead cells [23 (link)]. Samples with viability lower than 95% were excluded from the study. The remaining samples did not differ significantly in the percentage of viable cells.