Fragmentation of macrophages during isolation confounds analysis of single cell preparations from mouse hematopoietic tissues

Millard, S. M.; Heng, O.; Opperman, K. S.; Sehgal, A.; Irvine, K. M.; Kaur, S.; Summers, K. M.; Sandrock, C. J.; Wu, A. C.; Magor, G. W.; Batoon, L.; Perkins, A. C.; Noll, J. E.; Zannettino, A. C.; Sester, D. P.; Levesque, J.-P.; Hume, D. A.; Raggatt, L. J.; Pettit, A. R.

doi:10.1101/2021.04.28.441876

bioRxiv · 10.1101/2021.04.28.441876

Fragmentation of macrophages during isolation confounds analysis of single cell preparations from mouse hematopoietic tissues

Millard, S. M.·Heng, O.·Opperman, K. S.·Sehgal, A.·Irvine, K. M.·Kaur, S.·Summers, K. M.·Sandrock, C. J.·Wu, A. C.·Magor, G. W.·Batoon, L.·Perkins, A. C.·Noll, J. E.·Zannettino, A. C.·Sester, D. P.·Levesque, J.-P.·Hume, D. A.·Raggatt, L. J.·Pettit, A. R.

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Abstract

Mouse hematopoietic tissues contain abundant and heterogeneous populations of tissue-resident macrophages attributed trophic functions in control of immunity, hematopoiesis and bone homeostasis. A systematic strategy to characterise macrophage subsets in mouse bone marrow (BM), spleen and lymph node, unexpectedly revealed macrophage surface marker staining typically emanated from membrane-bound subcellular remnants associated with unrelated cell types. Remnant-restricted macrophage-specific membrane markers, cytoplasmic fluorescent reporters and mRNA were all detected in non-macrophage cell populations including isolated stem and progenitor cells. The profile of macrophage remnant association reflects adhesive interactions between macrophages and other cell types in vivo. Applying this knowledge, reduced macrophage remnant attachment to BM granulocytes in Siglec1 deficient mice was associated with compromised emergency granulocytosis, revealing a function for Siglec1-dependent granulocyte-macrophage interactions. Analysis of published RNA-seq data for purified macrophage and non-macrophage populations indicates that macrophage fragmentation is a general phenomenon that confounds bulk and single cell analysis of disaggregated tissues.

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Millard, S. M., Heng, O., Opperman, K. S., Sehgal, A., Irvine, K. M., Kaur, S., Summers, K. M., Sandrock, C. J., Wu, A. C., Magor, G. W., Batoon, L., Perkins, A. C., Noll, J. E., Zannettino, A. C., Sester, D. P., Levesque, J.-P., Hume, D. A., Raggatt, L. J., Pettit, A. R.. 2021-04-28. Fragmentation of macrophages during isolation confounds analysis of single cell preparations from mouse hematopoietic tissues. https://doi.org/10.1101/2021.04.28.441876

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