Flow cytometric gating for spleen monocyte and DC subsets: differences in autoimmune NOD mice and with acute inflammation.
The role of antigen presenting cells (APCs) in the pathogenesis of autoimmune and other inflammatory diseases is now better understood due to advances in multicolor flow cytometry, gene expression analysis of APC populations, and functional correlation of mouse to human APC populations. A simple but informative nomenclature of conventional and plasmacytoid dendritic cell subsets (cDC1, cDC2, pDC) and monocyte-derived populations incorporates these advances, but accurate subset identification is critical. Ambiguous gating schemes and alterations of cell surface markers in inflammatory condition can make comparing results between studies difficult. Both acute inflammation, such as TLR-ligand stimulation, and chronic inflammation as found in mouse models of autoimmunity can alter DC subset gating. Here, we address these issues using in vivo CpG stimulation as an example of acute inflammation and the non-obese diabetic (NOD) mouse as a model of chronic inflammation.We provide a flow cytometric antibody panel and gating scheme that differentiate 2 monocytic and 3DC subsets in the spleen both at steady state and after CpG stimulation. Using this method, we observed differences in the composition of NOD DCs that have been previously reported, and newly identified increases in the number of NOD monocyte-derived DCs. Finally, we established a protocol for DC phosphoflow to measure the phosphorylation state of intracellular proteins, and use it to confirm functional differences in the identified subsets. Therefore, we present optimized methods for distinguishing monocytic and DC populations with and without inflammation and/or autoimmunity associated with NOD mice.
Immune Tolerance Section, Diabetes, Endocrinology and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Matthew B
MB
Dong
Immune Tolerance Section, Diabetes, Endocrinology and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
M Jubayer
MJ
Rahman
Immune Tolerance Section, Diabetes, Endocrinology and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: tarbellk@niddk.nih.gov.
Kristin V
KV
Tarbell
D000208:Acute Disease; D000818:Animals; D001327:Autoimmune Diseases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015551:Autoimmunity; D015415:Biomarkers / Q000378:metabolism; D002469:Cell Separation / Q000379:methods*; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D004195:Disease Models, Animal; D005260:Female; D005434:Flow Cytometry; D016130:Immunophenotyping; D007249:Inflammation / Q000139:chemically induced / Q000276:immunology* / Q000378:metabolism; D007371:Interferon-gamma / Q000494:pharmacology; D008810:Mice, Inbred C57BL; D016688:Mice, Inbred NOD; D009000:Monocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D010641:Phenotype; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000276:immunology / Q000378:metabolism; D013154:Spleen / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D051217:Toll-Like Receptor 9 / Q000819:agonists
D015415:Biomarkers; C526902:CpG ODN 2216; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D051217:Toll-Like Receptor 9; D007371:Interferon-gamma
Dendritic cells; Immune homeostasis; Inflammation; Multicolor flow cytometry; Non-obese diabetic mice; Phosphoflow