Calcium phosphate coated 48 well plate

Murine BM cells were cultured for three days in complete MEM-α media supplemented with 50 ng/mL recombinant murine M-CSF and plated in a calcium phosphate-coated 48-well plate (Cosmo Bio USA, Carlsbad, CA, USA). Cells were cultured in complete MEM-α media containing both M-CSF (50 ng/mL) and RANKL (500 ng/mL), with vehicle (ethanol) or JTE013 (8 µM). A control group of cells was cultured with only M-CSF. On the third and fifth day, the cell culture media was changed with or without RANKL and/or JTE013. Some of the cells were stimulated with Aa-stimulated culture media (200 µL/mL) alone or co-stimulated with RANKL and Aa-stimulated culture media. Seven days after treatment, cells were removed by treatment with 5% sodium hypochlorite for 5 min. After washing and drying of the plate, bone resorption pit images were taken by a Nikon Eclipse TS-100 inverted microscope and analyzed by Visiopharm 5.0 software.

Murine BM cells were cultured for three days in complete MEM-α media supplemented with 50 ng/mL recombinant murine M-CSF and plated in a calcium phosphate-coated 48-well plate (Cosmo Bio USA, Carlsbad, CA). Cells were either untreated or treated with lentivirus containing either S1PR2 shRNA or control shRNA (moi 20). After lentiviral infection for 5 h, the cells were cultured in complete MEM-α media containing both M-CSF (50 ng/ml) and RANKL (500 ng/ml). A control group of cells were cultured with only M-CSF. On the 3^rd and 5^th day after lentiviral treatment, the cell culture media was changed with or without RANKL, and cells were either untreated or treated with Aa-stimulated media (100μL/well). Seven days after treatment, cells were removed by treatment with 5% sodium hypochlorite for 5 min. After washing and drying the plate, bone resorption pit images were taken by a Nikon Eclipse TS-100 inverted microscope and analyzed by Visiopharm 5.0 software.