pp 1-13 | Cite as
Isolation and Identification of Murine Bone Marrow-Derived Macrophages and Osteomacs from Neonatal and Adult Mice
Hematopoietic stem cells (HSCs) are regulated by multiple components of the hematopoietic niche, including bone marrow-derived macrophages and osteomacs. However, both macrophages and osteomacs are phenotypically similar. Thus, specific phenotypic markers are required to differentially identify the effects of osteomacs and bone marrow macrophages on different physiological processes, including hematopoiesis and bone remodeling. Here, we describe a protocol for isolation of murine bone marrow-derived macrophages and osteomacs from neonatal and adult mice and subsequent identification by multi-parametric flow cytometry using an 8-color antibody panel.
KeywordsBone digestion CD166 Hematopoietic niche Bone-marrow macrophages Multiparameter flow cytometry Osteomacs
The authors thank Indiana University Melvin and Bren Simon Cancer Center Flow Cytometry Resource Facility (FCRF) for their outstanding technical help and support. FCRF is partially funded by National Cancer Institute grant P30 CA082709 and National Institute of Diabetes and Digestive and Kidney Diseases grant U54 DK106846. We also thank the support of the NIH instrumentation grant 1S10D012270 for partial funding of the FCRF.
- 2.Ludin A, Itkin T, Gur-Cohen S, Mildner A, Shezen E, Golan K, Kollet O, Kalinkovich A, Porat Z, D'Uva G, Schajnovitz A, Voronov E, Brenner DA, Apte RN, Jung S, Lapidot T (2012) Monocytes-macrophages that express alpha-smooth muscle actin preserve primitive hematopoietic cells in the bone marrow. Nat Immunol 13(11):1072–1082. https://doi.org/10.1038/ni.2408CrossRefGoogle Scholar
- 3.Mohamad SF, Xu L, Ghosh J, Childress PJ, Abeysekera I, Himes ER, Wu H, Alvarez MB, Davis KM, Aguilar-Perez A, Hong JM, Bruzzaniti A, Kacena MA, Srour EF (2017) Osteomacs interact with megakaryocytes and osteoblasts to regulate murine hematopoietic stem cell function. Blood Adv 1(26):2520–2528. https://doi.org/10.1182/bloodadvances.2017011304CrossRefGoogle Scholar
- 4.Alexander KA, Chang MK, Maylin ER, Kohler T, Muller R, Wu AC, Van Rooijen N, Sweet MJ, Hume DA, Raggatt LJ, Pettit AR (2011) Osteal macrophages promote in vivo intramembranous bone healing in a mouse tibial injury model. J Bone Miner Res 26(7):1517–1532. https://doi.org/10.1002/jbmr.354CrossRefGoogle Scholar
- 5.Winkler IG, Sims NA, Pettit AR, Barbier V, Nowlan B, Helwani F, Poulton IJ, van Rooijen N, Alexander KA, Raggatt LJ, Levesque JP (2010) Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs. Blood 116(23):4815–4828. https://doi.org/10.1182/blood-2009-11-253534CrossRefGoogle Scholar
- 6.Bowen MA, Aruffo A (1999) Adhesion molecules, their receptors, and their regulation: analysis of CD6-activated leukocyte cell adhesion molecule (ALCAM/CD166) interactions. Transplant Proc 31(1–2):795–796Google Scholar
- 10.Chitteti BR, Kobayashi M, Cheng Y, Zhang H, Poteat BA, Broxmeyer HE, Pelus LM, Hanenberg H, Zollman A, Kamocka MM, Carlesso N, Cardoso AA, Kacena MA, Srour EF (2014) CD166 regulates human and murine hematopoietic stem cells and the hematopoietic niche. Blood 124(4):519–529. https://doi.org/10.1182/blood-2014-03-565721CrossRefGoogle Scholar