osteoblasts. Cells were incubated in different formulations and regimens of osteogenic medium as described in the following table (Table 3.1). Table 3.1: Different formulation and regimens of osteogenic medium Series 1st formulation and regimens 2nd formulation and regimens (Days 0-14) (Days 15-28) 1 medium + 10% FCS + 10 As 1st formulation α-MEM mM of + 50 β-glycerophosphate µg/ml of ascorbic acid. 2 medium + 10% FCS + 10 medium + 10% FCS + 10 α-MEM α-MEM mM of + 50 mM of + 50 β-glycerophosphate β-glycerophosphate µg/ml of ascorbic acid. µg/ml of ascorbic acid + 10-8 M of dexamethasone. 3 medium + 10% FCS + 10 As 1st formulation α-MEM mM of + 50 β-glycerophosphate µg/ml of ascorbic acid + 10-8 M of dexamethasone. 4 medium + 10% FCS, 10 As 1st formulation α-MEM mM of 50 β-glycerophosphate, µg/ml of ascorbic acid + H2O. Molecular biology RNA isolation and quantitation For osteoblasts, nearly confluent T25 flasks were washed twice with PBS and total RNA was extracted from primary osteoblasts at different stages of development using TriZol. For myeloma cell lines 5T33MM and 5TGM1 cells around 1x106 cells were washed twice with PBS and total RNA was extracted using TriZol. Total RNA was precipitated using isopropanol and re-suspended in RNase and DNase free H2O as described in section 2.2.5.1. NanoDrop ND 1000 Spectrophotometer (NanoDrop
osteoblasts. Cells were incubated in different formulations and regimens of osteogenic medium as described in the following table (Table 3.1). Table 3.1: Different formulation and regimens of osteogenic medium Series 1st formulation and regimens (Days 0-14) 2nd formulation and regimens (Days 15-28) 1 α-MEM medium + 10% FCS + 10 mM of β-glycerophosphate + 50 µg/ml of ascorbic acid. As 1st formulation 2 α-MEM medium + 10% FCS + 10 mM of β-glycerophosphate + 50 µg/ml of ascorbic acid. α-MEM medium + 10% FCS + 10 mM of β-glycerophosphate + 50 µg/ml of ascorbic acid + 10-8 M of dexamethasone. 3 α-MEM medium + 10% FCS + 10 mM of β-glycerophosphate + 50 µg/ml of ascorbic acid + 10-8 M of dexamethasone. As 1st formulation 4 α-MEM medium + 10% FCS, 10 mM of β-glycerophosphate, 50 µg/ml of ascorbic acid + H2O. As 1st formulation Molecular biology RNA isolation and quantitation For osteoblasts, nearly confluent T25 flasks were washed twice with PBS and total RNA was extracted from primary osteoblasts at different stages of development using TriZol. For myeloma cell lines 5T33MM and 5TGM1 cells around 1x106 cells were washed twice with PBS and total RNA was extracted using TriZol. Total RNA was precipitated using isopropanol and re-suspended in RNase and DNase free H2O as described in section 2.2.5.1. NanoDrop ND 1000 Spectrophotometer (NanoDrop
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