Chirayu Patel1, Lihong Shi1, John F Whitesides2,3, Brittni M Foster1, Roberto J Fajardo4, Ellen E Quillen5, Bethany A Kerr1,3,6. 1. Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina. 2. Department of Microbiology and Immunology, Wake Forest School of Medicine, Winston-Salem, North Carolina. 3. Wake Forest Baptist Comprehensive Cancer Center, Winston-Salem, North Carolina. 4. University of the Incarnate Word School of Osteopathic Medicine, San Antonio, Texas. 5. Department of Internal Medicine, Section on Molecular Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina. 6. Department of Orthopaedic Surgery, Wake Forest School of Medicine, Winston-Salem, North Carolina.
Abstract
The bone microenvironment cellular composition plays an essential role in bone health and is disrupted in bone pathologies, such as osteoporosis, osteoarthritis, and cancer. Flow cytometry protocols for hematopoietic stem cell lineages are well defined and well established. Additionally, a consensus for mesenchymal stem cell flow markers has been developed. However, flow cytometry markers for bone-residing cells-osteoblasts, osteoclasts, and osteocytes-have not been proposed. Here, we describe a novel partial digestion method to separate these cells from the bone matrix and present new markers for enumerating these cells by flow cytometry. We optimized bone digestion and analyzed markers across murine, nonhuman primate, and human bone. The isolation and staining protocols can be used with either cell sorting or flow cytometry. Our method allows for the enumeration and collection of hematopoietic and mesenchymal lineage cells in the bone microenvironment combined with bone-residing stromal cells. Thus, we have established a multi-fluorochrome bone marrow cell-typing methodology.
The bone microenvironment cellular composition plays an essential role in bone health and is disrupted in bone pathologies, such as osteoporosis, osteoarthritis, and cancer. Flow cytometry protocols for hematopoietic stem cell lineages are well defined and well established. Additionally, a consensus for mesenchymal stem cell flow markers has been developed. However, flow cytometry markers for bone-residing cells-osteoblasts, osteoclasts, and osteocytes-have not been proposed. Here, we describe a novel partial digestion method to separate these cells from the bone matrix and present new markers for enumerating these cells by flow cytometry. We optimized bone digestion and analyzed markers across murine, nonhuman primate, and human bone. The isolation and staining protocols can be used with either cell sorting or flow cytometry. Our method allows for the enumeration and collection of hematopoietic and mesenchymal lineage cells in the bone microenvironment combined with bone-residing stromal cells. Thus, we have established a multi-fluorochrome bone marrow cell-typing methodology.
Authors: Asha Shekaran; James T Shoemaker; Taylor E Kavanaugh; Angela S Lin; Michelle C LaPlaca; Yuhong Fan; Robert E Guldberg; Andrés J García Journal: Bone Date: 2014-08-27 Impact factor: 4.398
Authors: Nikolaos A Dallas; Shaija Samuel; Ling Xia; Fan Fan; Michael J Gray; Sherry J Lim; Lee M Ellis Journal: Clin Cancer Res Date: 2008-04-01 Impact factor: 12.531
Authors: Bridget E McLaughlin; Nicole Baumgarth; Martin Bigos; Mario Roederer; Stephen C De Rosa; John D Altman; Douglas F Nixon; Janet Ottinger; Carol Oxford; Thomas G Evans; David M Asmuth Journal: Cytometry A Date: 2008-05 Impact factor: 4.714
Authors: Brittni M Foster; Lihong Shi; Koran S Harris; Chirayu Patel; Victoria E Surratt; Kendall L Langsten; Bethany A Kerr Journal: Front Oncol Date: 2022-04-19 Impact factor: 5.738