Poster Presentation Australasian Cytometry Society 44th Annual Conference and Workshop

Insights into Flow Cytometric Data Analysis Discovered in a Quest to Isolate Ex Vivo Macrophages (24537)

Susan Millard 1 , Ostyn Heng 1 , Simranpreet Kaur 1 , David P Sester 2 , Jean-Pierre Levesque 1 , David A Hume 1 , Kim M Summers 1 , Allison R Pettit 1
  1. Mater Research Insitute - University of Queensland, Translational Research Institute, Woolloongabba, QLD, Australia
  2. TRI Flow Cytometry Suite, Translational Research Institute, Woolloongabba, QLD, Australia

Mouse hematopoietic tissues contain abundant tissue-resident macrophages that support immunity, hematopoiesis and bone homeostasis. A systematic strategy was applied to characterise macrophage subsets in mouse bone marrow (BM), spleen and lymph node. Imaging flow cytometry (IFC) revealed that none of the resultant candidate populations represented intact macrophages. Rather, macrophage surface marker expression emanated from, and was restricted to, membrane-bound subcellular remnants. Mapping macrophage marker expression across all hematopoietic cells demonstrated significant apparent expression across many non-macrophage cell types in BM, spleen and lymph node, with the phenomenon being most evident in BM. Enzymatic digestion was not sufficient to remove macrophage remnants from cells of other lineages in flushed BM. Recipient macrophages are resistant to myeloablative conditioning in transplantation models. IFC demonstrates that CD45.1/CD45.2 dual positive events in CD45.1 recipients of CD45.2 marrow are donor-derived cells carrying remnants of these resilient recipient macrophages. Remnant binding profiles differ between neonatal and adult BM, perhaps reflecting different cell niches.  Remnant contamination is not limited to hematopoietic tissues. Kupffer cell preparations isolated from liver using enzymatic digestion contain both intact cells and macrophage remnants adhered to other cells. Despite this prevalent artefact, conventional flow cytometry data can yield insights into macrophage biology. In naïve BM, CD4dim events were found to have a similar staining distribution to macrophage markers. IFC confirmed remnant-restricted CD4 staining. CD4 expression on murine BM macrophages is consistent with reports of a CD4-driven reporter marking a subset of perivascular macrophages in other murine tissues, and expression of CD4 on monocytes and macrophages in humans. It is not yet clear whether CD4 may distinguish different functional subsets of macrophages in the murine BM.