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Cytometry A. 2004 Oct;61(2):170-77.

Accurate and simple discrimination of mouse pulmonary dendritic cell and macrophage populations by flow cytometry: methodology and new insights.

Author information

1
Department of Respiratory Diseases, Ghent University Hospital 7K12ie, De Pintelaan 185, B-9000, Ghent, Belgium. Karim.Vermaelen@UGent.be

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.

PMID:
15382026
DOI:
10.1002/cyto.a.20064
[Indexed for MEDLINE]
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