Application of BioLegend’s Tools to Explore Bone Cell Differentiation and Bone Cancer
Mohar Chattopadhyay, PhD
Tuesday, December 12, 2017
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Chapters
Introduction
Agenda
Osteoclasts and Osteoblasts
Bone Marrow (BM) Cells Isolation Flowchart
Markers Commonly Expressed by BM Cells
BM Derived Macrophages (BMDM) Flowchart
BMDM Stain Positive for Macrophage Markers
BM Cell Differentiation Markers Staining Pattern
Phagocytosis Assay Protocol
BMDM Exhibit Phagocytosis
RANKL, RANK and OPG
BMDM to Osteoclast Differentiation Flowchart
rmRANKL Induces Osteoclast Differentiation
TRAP Activity Assay Protocol
Osteoclast Detection by TRAP Assay
Osteoclast Formation from Raw 264.7 Cells
Raw 264.7 Cells to Osteoclast Differentiation
mRANKL Induces Multinucleated Cells in a Dose Dependent Manner
rmRANKL Induces TRAP-Positive Cells in a Dose Dependent Manner
Osteoblasts
Osteoblast Differentiation Factors
Osteoblast Differentiation Assay Protocol
Recombinant Hedgehog Proteins Induce C3H10T1/2 Cell Differentiation to Osteoblasts
Recombinant PTH, DKK-1, Endoglin Proteins Modulate MC3T3-E1 Cell Differentiation to Osteoblasts
Part II. Normal Bone Homeostasis
RANKL- RANK Signaling
Nuclear Translocation of c-FOS and NFATc1
ChIP Assay Outline
ChIP with Anti-NF-kB and c-Fos Antibodies
ChIP with anti-NFATc1 Antibody (clone7A6)
PART III. Bone Cancer
Part III A - Bone Metastasis
Experimental Set-Up
rhRANKL Induces MDA-MB-231 Cell Invasion
rhOPG Inhibits rhRANKL Induced MDA-MB-231 Cell Invasion
RANKL- RANK Signaling
RANKL Activates MAPK Signaling in PC3 Cells
PART III B – Primary Bone Tumor Cells
Mitochondrial Morphology Changes
Conclusions
Acknowledgments
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