Fetal bovine serum fbs 16 000 044

The cell resuscitation process was described in previous papers [7 (link),14 (link)]. In brief, second-generation mammary epithelial cells were taken from liquid nitrogen and placed in a 37 °C water bath. Cells were centrifuged at 150× g for 5 min at 4 °C to isolate BMEC, and the precipitate rinsed with fresh medium prior to transferring to a new culture bottle. Once 90% confluence was reached, cultured cells were harvested with 0.25% trypsin EDTA and seeded at a density of 5 × 10⁴ cells/mL into 6-well plates (3335, Corning Life Science, New York, NY, USA), containing growth medium (DMEM/F12 with 10% Fetal bovine serum (FBS (16000-044, Gibco, Carlsbad, CA, USA)), 500 ng/mL hydrocortisone (S0135, Sigma, Saint Louis, MI, USA), 1 µg/L prolactin (L6520, Sigma, Saint Louis, MI, USA), 10 ng/mL epidermal growth factor (AF-100-15, Peprotech, Rocky Hill, CT, USa), and 100 IU/mL penicillin/streptomycin)). Each treatment was replicated 6 times and cultured in an incubator at 37 °C, 95% O₂, and 5% CO₂. Cells were incubated with DMEM/F12 medium without fetal bovine serum for 16 h, to eliminate the effects from serum, then the cells were incubated in medium with different treatments for another 24 h.

Minimum Essential Medium with alpha modifications (α-MEM, #LM008-01) and Dulbecco’s phosphate-buffered saline (DPBS, #LB001-01) were purchased from WelGENE Inc. (Daegu, Korea). Fetal bovine serum (FBS, #16000044), trypsin-EDTA (#25200056), and penicillin-streptomycin (#15140122) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Recombinant human TNFα (#300-01A), IL-1β (#200-01B), and M-CSF (#300–25) were purchased from Peprotech (Rocky Hill, NJ, USA). Recombinant mouse RANKL (#cyt-320) was purchased from Prospec (Israel). Anti-cathepsin K (#ab19027) antibody was purchased from Abcam (Cambridge, MA, USA). TRAP staining kit (#387A) was purchased from Sigma (St. Louis, MO, USA). Culture plates were purchased from Nunc (Roskilde, Denmark), and Transwell (#37024) was purchased from SPL (Pocheon, Korea).