Technical Reference #1730

1730.

Breast Cancer-derived Factors Stimulate Osteoclastogenesis through the Ca2+/Protein Kinase C and Transforming Growth Factor-B/MAPK Signaling Pathways Kerstin Tiedemann; Osama Hussein; Gulzhakhan Sadvakassova; Yubin Guo; Peter M. Siegel; Svetlana V. Komarova, McGill University, Journal of Biological Chemistry, 284(1730), (2009)
Link To Paper

Materials & Methods:
MDA-MB-231 and MCF10a cells were kindly provided by Dr. J. Massague´ (Memorial Sloan-Kettering Cancer Center New York). MDA-MB-231 were cultured in the incubation medium (DMEM with 1.5 g/liter sodium bicarbonate glutamine (319-020-CL Wisent Inc.) 1mM pyruvate (600-110-EL Wisent Inc.) 100 units/ml penicillin 100 g/ml streptomycin (450-201-EL Wisent Inc.) 10% fetal bovine serum (080-150 Wisent Inc.)) and conditioned media (CM) was harvested after 24-h incubation. MCF10a were cultured to confluence in a 1:1 mixture of incubation medium and Ham’s F-12 (Amersham Biosciences/Invitrogen) with 10% fetal calf serum 10 g/ml insulin (Sigma) 0.5 g/ml hydrocortisone (Sigma) 0.02 g/m epidermal growth factor (Sigma) and CM was harvested 24 h later. MCF10a culture medium does not affect osteoclast formation (12). CM was filtered through 0.22 m aliquoted and frozen at 80 °C.

Microscopic Technique
Fluorescence inverted microscope

Cell Type(s)
MDA-MB-231 & MCF10a cells; RAW 264.7 cells