ArticleJournal of immunological methods2012
Autofluorescence contributes to false-positive intracellular Foxp3 staining in macrophages: a lesson learned from flow cytometry.
Article in Journal of immunological methods, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 30 papers.
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Who cites it
30 citing papers in PubMed, 67 citations in OpenAlex.
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- Breast Cancer Cell Re-Dissemination from Lung Metastases-A Mechanism for Enhancing Metastatic Burden.Journal of clinical medicine · 2021Article
- AutoSpill is a principled framework that simplifies the analysis of multichromatic flow cytometry data.Nature communications · 2021Article
- Identification of PD-1 ligands: PD-L1 and PD-L2 on macrophages in lung cancer milieu by flow cytometry.Translational lung cancer research · 2021Article
- Corrected Super-Resolution Microscopy Enables Nanoscale Imaging of Autofluorescent Lung Macrophages.Biophysical journal · 2020Article
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- Multiplex Immunofluorescence of Bone Marrow Core Biopsies: Visualizing the Bone Marrow Immune Contexture.The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society · 2020Article
- Immune contexture analysis in immuno-oncology: applications and challenges of multiplex fluorescent immunohistochemistry.Clinical & translational immunology · 2020Review
- Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).European journal of immunology · 2019Article
- Fluorescence lifetime shifts of NAD(P)H during apoptosis measured by time-resolved flow cytometry.Cytometry. Part A : the journal of the International Society for Analytical Cytology · 2019Article
- Deficiency of GATA3-Positive Macrophages Improves Cardiac Function Following Myocardial Infarction or Pressure Overload Hypertrophy.Journal of the American College of Cardiology · 2018Article
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Authors and funding
8 authors at 1 institution in 1 country.
Funding
Abstract
Forkhead box P3 (Foxp3) is well known for its highly restricted expression in T regulatory cells (Tregs). A recent study suggested the existence of a Foxp3 positive macrophage subpopulation in mouse bone marrow, spleen, liver, lymph nodes, and thymus that exhibited immune regulatory effect similar to Tregs. Before this report was retracted, we attempted to study the function of this macrophage subpopulation in a mouse model of hyperlipidemia. Bone marrow and spleen cells isolated from C57BL/6 apo E(-/-) mice were stained with anti-CD11b, anti-F4/80 and anti-Foxp3 and analyzed by flow cytometry. Our results showed that 3.06-8.08% of CD11b(+)F4/80(+) macrophages from bone marrow cells and 0.24-2.21% from splenic were Foxp3-positive. Unexpectedly, unstained or isotype stained controls also showed strong autofluorescence and similar percentages of these cells fell within the same FL1 channel that counted the anti-Foxp3 stained population. Back gating of the autofluorescent population onto a SSC/FSC plot showed that this population of cells had a higher side scatter. The peritoneal macrophages (PMø) exhibited similar autofluorescence. We used qPCR to further evaluate the expression of Foxp3 mRNA in PMø that were treated with M-CSF, M-CSF+IL-4, M-CSF+TGFβ1 or in BMDM treated with TGFβ1 in the presence of anti-CD3 and CD28 antibody co-stimulators. No expression of Foxp3 mRNA was detected in either cell culture systems, whereas robust Foxp3 gene expression was induced in naïve CD4+ cells stimulated with TGFβ1. Consistent with these findings, fluorescence microscopy showed no Foxp3 protein expression in PMø, however Foxp3 expression was easily detected in induced Tregs. We conclude that the reported expression of Foxp3 in macrophages is likely an artifact and that a stringent multimodality approach is critical to demonstrate candidate gene expression in any cell type.
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