Minimum Essential Medium (MEM)

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	supplemented with 10% SERA Plus specially processed fetal bovine serum (FBS) (PAN biotech GmbH), penicillin (100 U/mL), streptomycin (100 μg/mL), and L-glutamine (2 mM).

Protocol tips
supplemented with 10% SERA Plus specially processed fetal bovine serum (FBS) (PAN biotech GmbH), penicillin (100 U/mL), streptomycin (100 μg/mL), and L-glutamine (2 mM).

Publication protocol

Primary cultures of human dermal fibroblasts (HDF) were purchased from Istituto Zooprofilattico Sperimentale, Brescia, Italy, and cultured in Minimum Essential Medium (MEM) (GIBCO) supplemented with 10% SERA Plus specially processed fetal bovine serum (FBS) (PAN biotech GmbH), penicillin (100 U/mL), streptomycin (100 μg/mL), and L-glutamine (2 mM). Upon thawing, a batch of cells was divided into two 25 cm2 flasks for two distinct culture conditions—one for cultures under standard atmospheric oxygen tension (21% O2) using an Heraeus BB15 incubator (Thermo Scientific, Germany) and the other for cultures under low oxygen tension (5% O2) using an HERAcell 150i incubator (Thermo Scientific, Germany) equipped with an oxygen sensor and nitrogen supply. Both incubators were maintained at 37°C and 5% CO2 under a humidified atmosphere. For the experiments, cells grown under the two oxygen tensions were seeded at an optimal density of 10 × 103 cell/cm2. Cell culture medium was changed every 2-3 days and fibroblasts were passaged at 80% confluence by trypsinization. Cells were serially cultured under their respective oxygen tension and expanded until enough cells were obtained for all experiments. For the experiments, the cells considered as young (Y) were those collected at the 10th passage (p) whereas those considered as old (O) were collected at the 24th p. The cells were always subcultured to maintain equivalent passage numbers.

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