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 CD8α. 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. © 2004 Wiley‐Liss, Inc.