Heat inactivated bovine serum

Primary sebocyte cultures were prepared from occipital hair follicle sebaceous glands. The sebaceous glands were isolated from hair follicles under a binocular microscope and transferred to a tissue culture dish. The cells were maintained in Dulbecco's modified Eagle medium (DMEM; Hyclone Laboratories, Logan, UT, USA) at 37℃ in a humidified atmosphere of 5% CO₂. Explants were left for a period of 4 days, and the medium was then changed to Epilife (MEPI500CA; Gibco BRL, Grand Island, NY, USA). The medium was changed every 2 or 3 days. DMEM was supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and 20% heat-inactivated bovine serum (Hyclone Laboratories), while Epilife was supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and fungizone (0.25 µg/ml).
Once the outgrowth cells became subconfluent, the cells were harvested using 0.25% trypsin and 10 mM ethylenediamine tetraacetic acid in Hank's balanced salt solution, followed by subculturing. The cells obtained after the second passage were used in this study after identifying the cultured sebocytes using Oil Red O (Sigma, St. Louis, MO, USA) staining and immunocytofluorescence against cytokeratins 1 and 7 (Chemicon, Billerica, MA, USA).

Primary sebocyte cultures were obtained from occipital hair follicle sebaceous glands. From the hair follicles, the sebaceous glands were dissected under a binocular microscope and transferred to a tissue culture dish. The cells were maintained in Dulbecco's modified Eagle medium (DMEM; Hyclone Laboratories, Logan, UT, USA) at 37℃ in a humidified atmosphere of 5% CO₂. Explants were incubated for three days, and the medium was then changed to Epilife (MEPI500CA; Gibco BRL, Grand Island, NY, USA). The medium was changed every three days. DMEM was supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and 20% heat inactivated bovine serum (Hyclone Laboratories), while Epilife was supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and fungizone (250 µg/ml).
Once the cells became subconfluent, they were harvested using 0.25% trypsin and 10 mM ethylenediamine tetraacetic acid (EDTA) in Hank's balanced salt solution, followed by subculturing at a split ratio of 1:3. Cells obtained after the second passage were used in this study. These cultured sebocytes were subjected to hematoxylin and eosin (Muto Pure Chemicals Co., Tokyo, Japan) and Oil Red O (Sigma, St. Louis, MO, USA) staining, and immunocytofluorescence against cytokeratin 1 and 7 (Chemicon, Billerica, MA, USA) before experimental use (Fig. 1).