Open AccessOsteoblast Sorting and Intracellular Staining of CXCL12
Author(s) -
Weihuan Wang,
Gurnoor Majihail,
Lui Cui,
Lan Zhou
Publication year - 2018
Publication title -
bio-protocol
Language(s) - English
Resource type - Journals
ISSN - 2331-8325
DOI - 10.21769/bioprotoc.2858
Subject(s) - stromal cell , bone marrow , microbiology and biotechnology , osteoblast , immunophenotyping , chemistry , haematopoiesis , stem cell , chemokine , cxcr4 , biology , flow cytometry , immunology , cancer research , receptor , in vitro , biochemistry
Osteoblasts are bone marrow endosteum-lining niche cells playing important roles in the regulation of hematopoietic stem cells by secreting factors and cell adhesion molecules. Characterization of primary osteoblasts has been achieved through culture of outgrowth of collagenase treated bone. Immunophenotyping and flow-based analysis of long bone osteoblasts offer a simplified and rapid approach to characterize osteoblasts. We describe a modified procedure of isolating mouse bone marrow osteoblastic cells based on cell surface immunophenotyping. The chemokine CXCL12 (also known as stromal-derived factor, SDF-1) together with its receptor CXCR4 are expressed by osteoblasts and bone marrow stroma cells. The CXCL12-CXCR4 axis is important for hematopoietic stem cell retention to their niches (Sugiyama et al. , 2006) and for supporting leukemia initiating cell activity (Pitt et al. , 2015). Here we describe the procedure of intracellular staining of CXCL12.