Isolation and Identification of Murine Bone Marrow-Derived Macrophages and Osteomacs from Neonatal and Adult Mice

  • Joydeep GhoshEmail author
  • Safa F. Mohamad
  • Edward F. Srour
Part of the Methods in Molecular Biology book series (MIMB, volume 2002)


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.


Bone 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.


  1. 1.
    Ginhoux F, Schultze JL, Murray PJ, Ochando J, Biswas SK (2016) New insights into the multidimensional concept of macrophage ontogeny, activation and function. Nat Immunol 17(1):34–40. CrossRefPubMedGoogle Scholar
  2. 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. CrossRefPubMedGoogle Scholar
  3. 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. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 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. CrossRefPubMedGoogle Scholar
  5. 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. CrossRefGoogle Scholar
  6. 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):795–796CrossRefGoogle Scholar
  7. 7.
    Chitteti BR, Bethel M, Kacena MA, Srour EF (2013) CD166 and regulation of hematopoiesis. Curr Opin Hematol 20(4):273–280. CrossRefPubMedGoogle Scholar
  8. 8.
    Swart GW (2002) Activated leukocyte cell adhesion molecule (CD166/ALCAM): developmental and mechanistic aspects of cell clustering and cell migration. Eur J Cell Biol 81(6):313–321. CrossRefPubMedGoogle Scholar
  9. 9.
    Chitteti BR, Cheng YH, Kacena MA, Srour EF (2013) Hierarchical organization of osteoblasts reveals the significant role of CD166 in hematopoietic stem cell maintenance and function. Bone 54(1):58–67. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 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. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Mair F, Prlic M (2018) OMIP-044: 28-color immunophenotyping of the human dendritic cell compartment. Cytometry A 93(4):402–405. CrossRefPubMedGoogle Scholar
  12. 12.
    Staser KW, Eades W, Choi J, Karpova D, DiPersio JF (2018) OMIP-042: 21-color flow cytometry to comprehensively immunophenotype major lymphocyte and myeloid subsets in human peripheral blood. Cytometry A 93(2):186–189. CrossRefPubMedGoogle Scholar
  13. 13.
    Autengruber A, Gereke M, Hansen G, Hennig C, Bruder D (2012) Impact of enzymatic tissue disintegration on the level of surface molecule expression and immune cell function. Eur J Microbiol Immunol (Bp) 2(2):112–120. CrossRefGoogle Scholar
  14. 14.
    McCabe A, MacNamara KC (2016) Macrophages: key regulators of steady-state and demand-adapted hematopoiesis. Exp Hematol 44(4):213–222. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Wu AC, Raggatt LJ, Alexander KA, Pettit AR (2013) Unraveling macrophage contributions to bone repair. Bonekey Rep 2:373. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2018

Authors and Affiliations

  • Joydeep Ghosh
    • 1
    Email author
  • Safa F. Mohamad
    • 2
  • Edward F. Srour
    • 1
    • 3
  1. 1.Department of MedicineIndiana University School of MedicineIndianapolisUSA
  2. 2.Department of Microbiology and ImmunologyIndiana University School of MedicineIndianapolisUSA
  3. 3.Department of Microbiology and ImmunologyIndiana University School of MedicineIndianapolisUSA

Personalised recommendations