Abstract
The erythroblastic island (EBI) is a multicellular functional erythropoietic unit comprising a central macrophage nurturing a rosette of maturing erythroblasts. Since the discovery of EBIs more than half a century ago, EBIs are still studied by traditional microscopy methods after enrichment by sedimentation. These isolation methods are not quantitative and do not enable precise quantification of EBI numbers or frequency in the bone marrow or spleen tissues. Conventional flow cytometric methods have enabled quantification of cell aggregates co-expressing macrophage and erythroblast markers; however, it is unknown whether these aggregates contain EBIs as these aggregates cannot be visually assessed for EBI content. Combining the strengths of both microscopy and flow cytometry methods, in this chapter we describe an imaging flow cytometry method to analyze and quantitatively measure EBIs from the mouse bone marrow. This method is adaptable to other tissues such as the spleen or to other species provided that fluorescent antibodies specific to macrophages and erythroblasts are available.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Erbani J, Tay J, Barbier V, Levesque JP, Winkler IG (2020) Acute myeloid leukemia chemo-resistance is mediated by E-selectin receptor CD162 in bone marrow niches. Front Cell Dev Biol 8:668. https://doi.org/10.3389/fcell.2020.00668
Gülcüler Balta GS, Monzel C, Kleber S, Beaudouin J, Balta E, Kaindl T et al (2019) 3D cellular architecture modulates tyrosine kinase activity, thereby switching CD95-mediated apoptosis to survival. Cell Rep 29:2295–2306.e2296. https://doi.org/10.1016/j.celrep.2019.10.054
Hritzo MK, Courneya J-P, Golding A (2018) Imaging flow cytometry: a method for examining dynamic native FOXO1 localization in human lymphocytes. J Immunol Methods 454:59–70. https://doi.org/10.1016/j.jim.2018.01.001
Gautam N, Sankaran S, Yason JA, Tan KSW, Gascoigne NRJ (2018) A high content imaging flow cytometry approach to study mitochondria in T cells: MitoTracker Green FM dye concentration optimization. Methods 134-135:11–19. https://doi.org/10.1016/j.ymeth.2017.11.015
Cerveira J, Begum J, Di Marco Barros R, van der Veen AG, Filby A (2015) An imaging flow cytometry-based approach to measuring the spatiotemporal calcium mobilisation in activated T cells. J Immunol Methods 423:120–130. https://doi.org/10.1016/j.jim.2015.04.030
Crane GM, Jeffery E, Morrison SJ (2017) Adult haematopoietic stem cell niches. Nat Rev Immunol 17:573. https://doi.org/10.1038/nri.2017.53
Levesque J-P, Jacobsen RN, Winkler IG (2017) The role of mesenchymal stem cells in hematopoiesis. In: Atkinson K (ed) The biology and therapeutic application of mesenchymal cells. Wiley, Hoboken, pp 467–480. https://doi.org/10.1002/9781118907474.ch32
Seu KG, Papoin J, Fessler R, Hom J, Huang G, Mohandas N et al (2017) Unraveling macrophage heterogeneity in Erythroblastic Islands. Front Immunol 8:1140. https://doi.org/10.3389/fimmu.2017.01140
Tay J, Bisht K, McGirr C, Millard SM, Pettit AR, Winkler IG et al (2020) Imaging flow cytometry reveals that granulocyte colony-stimulating factor treatment causes loss of erythroblastic islands in the mouse bone marrow. Exp Hematol 82:33–42. https://doi.org/10.1016/j.exphem.2020.02.003
Bessis M (1958) L'îlot érythroblastique. Unité fonctionnelle de la moelle osseuse. Rev Hematol 13:8–11
Jacobsen RN, Perkins AC, Levesque JP (2015) Macrophages and regulation of erythropoiesis. Curr Opin Hematol 22:212–219. https://doi.org/10.1097/moh.0000000000000131
Yeo JH, Lam YW, Fraser ST (2019) Cellular dynamics of mammalian red blood cell production in the erythroblastic island niche. Biophys Rev. https://doi.org/10.1007/s12551-019-00579-2
Li W, Wang Y, Zhao H, Zhang H, Xu Y, Wang S et al (2019) Identification and transcriptome analysis of erythroblastic island macrophages. Blood 134:480–491. https://doi.org/10.1182/blood.2019000430
Bisht K, Tay J, Wellburn RN, McGirr C, Fleming W, Nowlan B et al (2020) Bacterial lipopolysaccharides suppress Erythroblastic Islands and erythropoiesis in the bone marrow in an extrinsic and G- CSF-, IL-1-, and TNF-independent manner. Front Immunol 11:2548. https://doi.org/10.3389/fimmu.2020.583550
Soni S, Bala S, Gwynn B, Sahr KE, Peters LL, Hanspal M (2006) Absence of erythroblast macrophage protein (Emp) leads to failure of erythroblast nuclear extrusion. J Biol Chem 281:20181–20189. https://doi.org/10.1074/jbc.M603226200
Lee SH, Crocker PR, Westaby S, Key N, Mason DY, Gordon S et al (1988) Isolation and immunocytochemical characterization of human bone marrow stromal macrophages in hemopoietic clusters. J Exp Med 168:1193–1198. https://doi.org/10.1084/jem.168.3.1193
Acknowledgments
The authors thank the Translational Research Institute, The University of Queensland, and Mater Foundation for providing an excellent research environment and Flow Core Facility. The development of this methodology was supported by funding from the National Health and Medical Research Council Research Fellowship 1136130 (J.P.L.), and Mater Foundation (J.P.L.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Tay, J., Bisht, K., Winkler, I.G., Levesque, JP. (2023). Imaging Flow Cytometric Analysis of Primary Bone Marrow Erythroblastic Islands. In: Barteneva, N.S., Vorobjev, I.A. (eds) Spectral and Imaging Cytometry. Methods in Molecular Biology, vol 2635. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3020-4_3
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3020-4_3
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3019-8
Online ISBN: 978-1-0716-3020-4
eBook Packages: Springer Protocols