Millicell cell culture plate insert

For the RNA and protein analyses, C3H10T1/2‐EV (empty virus) and C3H10T1/2‐Wnt1 cells were seeded in 12‐well plates at the density of 40,000 cells per well. For mixed cultures, 10T1/2‐EV and 10T1/2‐Wnt1 cells were seeded in 12‐well plates at equal amounts of 20,000 and 20,000 cells, respectively. For insert culture, 10T1/2‐EV and 10T1/2‐Wnt1 cells were seeded onto a Millicell cell culture plate insert (Millipore) at a density of 10,000 per well. Millicell inserts were placed in 12‐well plates and 40,000 C3H10T1/2‐EV cells were seeded in the lower chambers. ALP staining, RNA, and protein extraction were performed at 10 days.

The osteoblastic cells (MC3T3‐E1, C3H10T1/2, primary calvarial osteoblast from WT or Wnt1^+/– mice) were plated on 24‐well plates at a density of 10,000 cells/well in α‐MEM with 10% FBS. Next day, nonadherent cells harvested from WT C57Bl/6N mouse bone marrow were plated on top of osteoblastic cells at a density of 1.5 × 10⁶/well (on the top of MC3T3‐E1 and primary cells) or 6.6 × 10⁴ (on top of C3H10T1/2 cells). The cells were stimulated either with 10^–8M vitamin D and 10^–6M prostaglandin E2 (MC3T3‐E1 cells, primary cells) or with 10 ng/mL M‐CSF and 50 ng/mL hRANKL (C3H10T1/2). The culture medium was changed every 3 days and TRAP staining was performed at indicated time points. For low‐density versus high‐density experiments, 1 × 0.1 and 1 × 10⁴ C3H10T1/2 cells /well were plated on 48‐well plates, respectively. To study osteoblast‐osteoclast interaction mediated by secreted factors, the co‐culture was performed by plating the nonadherent bone marrow cells on the 12‐well plate at a density of 1.32 ×10⁵/well and C3H10T1/2‐EV/Wnt1 cells onto a Millicell cell culture plate insert (Millipore) at a density of 5800 per well.