2.2 Cell Culture Method
Murine preosteoblastic cells MC3T3-E1 (ATCC CRL-2593) were obtained and expanded according to established protocols.22, 23 Cells were seeded on T-150 flasks (Corning) at 5000 cells/cm2 and expanded in a growth media consisting of MEM ⍺ with nucleosides (Gibco) with 10% fetal bovine serum (Atlanta Biologicals, Lot #E15052) and 1% penicillin/streptomycin (Corning). Media was replenished with clean growth media every 2-3 days until cells reached 80% confluence. Once confluent, cells were washed with phosphate-buffered saline (PBS, Gibco) and detached from flasks with 0.25% trypsin-EDTA (Gibco). Dissociated cells were counted and replated onto 6-well plates at 5x105 per well (53,000 cells/cm2). After overnight adhesion, MC3T3s were separated into two groups, one group subjected to osteogenic differentiation (i.e., differentiated group) using the In Vitro Osteogenesis Assay Kit (ECM810, EMD Millipore) and the other cultured in growth media (i.e., undifferentiated group). For cells undergoing osteogenic differentiation, media was aspirated and replaced with 2.5 mL growth media supplemented with 0.2 mM ascorbic acid 2-phosphate (EMD Millipore), and 10 mM glycerol 2-phosphate (EMD Millipore) while non-differentiated cells were aspirated and given 2.5 mL growth media with no additives. Every 3 days, media was replaced after DSP sampling. Immediately after samples of conditioned media from both differentiated and non-differentiated MC3T3s were collected, the media was replaced. After 6 days in culture, the differentiation media was also supplemented with 50 nM melatonin (EMD Millipore). Both cell groups were cultured for 18 days total, being sampled 6 times total. After the final DSP experiment (day 18) cells were washed with PBS and fixed with 4% paraformaldehyde (Sigma Aldrich) in PBS at room temperature for 15 minutes. Fixative was removed and the cells were carefully rinsed three times with distilled water. Next, differentiated and undifferentiated MC3T3s were incubated in 2 mL alizarin red staining solution (EMD Millipore) for 20 minutes. Staining solution was then removed and cells were washed 4 times with deionized water. Osteogenic differentiation was confirmed in the differentiated group by red staining of calcium deposition, while no staining was observed in the non-differentiated group, as expected (Fig. S7).