Penicillin and streptomycin pen strep

Human adrenocortical NCI-H295R (NCI-H295R) cells were obtained from American Type Culture Collection (ATCC-LGC Standards GmbH, Wesel, Germany). Cells were cultured under standard conditions in Dulbecco's modified Eagle's/Ham's F-12 medium (DMEM/F12; Gibco, Life Technologies Europe BV, Bleiswijk, The Netherlands) supplemented with 2.5% Nu-serum (BD Biosciences, Breda, The Netherlands), 1% insulin/transferrin/selenium (ITS, BD Biosciences), and 1% penicillin and streptomycin (pen/strep, Gibco, Life Technologies Europe BV). Other biochemical reagents, including metformin and pioglitazone, were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.

Human ASCs were isolated from lipoaspirate, and were characterized and expanded according to protocols described by Zuk (2001 ) and Bunnell et al. (2008 (link); Zuk 2001 ). In short, lipoaspirate was washed three times with phosphate-buffered saline (PBS) to remove contaminating peripheral blood cells, after which the sample was subjected to enzymatic digestion to separate the stromal vascular fraction (SVF) from mature adipocytes. The red blood cells were lysed and the cell suspension strained through a 70 µm cell strainer (Millipore, Billerica, MA, USA) before seeding the SVF into culture flasks at a density of 5.0 × 10⁵ cells/cm². After 24 h the non-adherent cells were removed resulting in selection for the adherent cell component present in the adipose derived SVF. ASC cultures were maintained under standard culture conditions (37 °C, 5 % CO₂) in alpha-Modified Eagle Medium GlutaMax™ culture medium (α-MEM, Gibco, Life Technologies/Thermo Fischer Scientific, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin and streptomycin (pen/strep, Gibco). At 80 % confluence, the cultures were passaged using 0.25 % Trypsin/EDTA (Gibco) and re-seeded at a density of 5.0 × 10³ cells/cm².

U2OS cells (ATCC HTB-96) were grown in DMEM (Lonza) supplemented with 10% fetal calf serum (Sigma), 2 mM glutamine and 1% penicillin and streptomycin (pen/strep) (Gibco). One day before fixation and immunostaining, cells were plated on 18-mm coverslips and grown to ~ 50% confluency.