title: Accurate and simple discrimination of mouse pulmonary dendritic cell and macrophage populations by flow cytometry: methodology and new insights.
abstract: BACKGROUND
  The need to accurately discriminate dendritic cells (DCs) and macrophages (Mphs) in mouse lungs is critical given important biological differences. However, a validated flow cytometry-based method is still lacking, resulting in much confusion between both cell types.
  
  
  METHODS
  Single-cell suspensions freshly obtained from collagenase-digested lung tissue were stained with a CD11c-specific monoclonal antibody, detected using a PE-Cy5 or APC-conjugated secondary reagent. Cellular immunophenotype was simultaneously explored using a panel of PE-conjugated markers. The FL1 or FITC-detection channel was reserved for the assessment of autofluorescence.
  
  
  RESULTS
  CD11c-bright cells were heterogeneous and displayed a bimodal distribution with regard to autofluorescence (AF). CD11c+/low-AF cells were lineage-negative and showed features compatible with myeloid DCs. This was confirmed by morphology, potent T-cell stimulatory function in a mixed-leukocyte reaction, surface expression of MHCII and costimulatory molecules, and further immunophenotypical criteria, including the expression of Mac-1 and absence of CD8alpha. In contrast, CD11c+/high-AF cells displayed the features of pulmonary Mphs, including typical Mph morphology, very weak induction of T-cell proliferation, low to absent expression of MHCII and costimulatory molecules, and very low levels of Mac-1 as well as F4/80. We also show that only CD11c+/high-AF cells strongly expressed the macrophage marker MOMA-2, while interestingly Mac-3 was expressed at high levels by CD11c+/high-AF and low-AF alike.
  
  
  CONCLUSIONS
  This study shows that the combination of CD11c-expression and autofluorescence is necessary and sufficient to accurately separate DCs from macrophage subpopulations in mouse lungs.
authors.affiliation: Department of Respiratory Diseases, Ghent University Hospital 7K12ie, De Pintelaan 185, B-9000, Ghent, Belgium. Karim.Vermaelen@UGent.be
authors.forename: Karim
authors.initials: K
authors.lastname: Vermaelen
authors.forename: Romain
authors.initials: R
authors.lastname: Pauwels
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000943:Antigens, Differentiation / Q000096:biosynthesis; D039521:CD11c Antigen / Q000096:biosynthesis; D016827:CD8 Antigens / Q000096:biosynthesis; D017364:Collagenases / Q000378:metabolism; D003713:Dendritic Cells / Q000166:cytology*; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D016130:Immunophenotyping; D007962:Leukocytes / Q000166:cytology; D008168:Lung / Q000166:cytology; D008264:Macrophages / Q000166:cytology* / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000943:Antigens, Differentiation; D039521:CD11c Antigen; D016827:CD8 Antigens; C036763:monocyte-macrophage differentiation antigen; D017364:Collagenases