Mc3t3 e1 subclone 4

Human PCa cell line PC-3 [American Type Culture Collection (ATCC), Manassas, VA, Cat #: CRL-1435] and DU145 cells (ATCC, Cat #: HTB-81) were transformed to stably express green fluorescent protein (GFP) and firefly luciferase (PC3-GFP-luc, and DU145-GFP-luc) by transduction with a lentivirus (Lenti-GF1-CMV-VSVG) generated by the University of Michigan Vector Core. The transduced cells were sorted for GFP positive cells at the Wake Forest Baptist Comprehensive Cancer Center Flow Cytometry Shared Resource using an Astrios EQ (Beckman Coulter, Pasadena, CA), expanded and frozen at low passage (<10). The growth media for PC-3 and DU145 was RPMI 1640 (Gibco, Gaithersburg, MD, Cat #: 11875093) supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO, Cat #: F2442), 1% Penicillin-Streptomycin (Gibco, Cat #: 15140122), and 1% L-Glutamine (Gibco, Cat #: 25030081). Murine calvarial pre-osteoblast cell line MC3T3-E1 Subclone 4 (ATCC, Cat #: CRL-2593) were cultured with MEMα without nucleosides (Gibco, Cat #: 12561056) supplemented with 10% FBS, 1% Penicillin-Streptomycin, and 1% L-Glutamine. Before supplementation, this formulation of MEMα already contains 50 mg/L Ascorbic Acid, and therefore no additional Ascorbic Acid was required. Cells were incubated at 37°C, 5% CO₂, and 100% humidity and were routinely passaged when no more than 80% confluent.

We commercially purchased MC3T3-E1 cell (MC3T3-E1 Subclone 4, ATCC CRL2593, Rockville, MD, USA) from American Type Culture Collection (Manassas, VA, USA). Compared with primary cell cultures, the clonal mouse pre-osteoblastic MC3T3-E1 cell line exhibits high levels of cellular differentiation and clear reproducibility.13 (link) MC3T3-E1 cells are similar to osteoblasts.14 (link) In particular, the MC3T3-E1 Subclone 4 cells represent a good model for studying in vitro osteoblast differentiation.13 (link) In addition, because of behavior similar to primary calvarial osteoblasts, they were considered appropriate for studying the impact of the implant surface on osteoblasts.13 (link) The osteoblastic cell line MC3T3-E1 was dispensed into α-Minimum Essential Medium (α-MEM, Dulbecco’s Modified Eagle Medium, Gibco-BRL, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) and 100 U/mL penicillin at a concentration of 1 x 10⁶ cells/mL, and was cultured in a carbon dioxide incubator set at 5% CO₂ and 37°C (FormaSeries II 3111 Water Jacketed CO₂ Incubator, Thermo Scientific, Waltham, MA, USA). The cell culture was performed in cell culture dishes (9 x 20 mm). The culture medium was changed every 3 days, and the MC3T3-E1 cells were subcultured 4 times.