The Identification and Enumeration of Dendritic Cell Populations from Individual Mouse Spleen and Peyer's Patches Using Flow Cytometric Analysis

Dendritic cell (DC) research currently involves pooling of tissues from multiple animals followed by enrichment techniques to obtain sufficient numbers of DCs for analysis. Enrichment techniques take advantage of DC adherence, buoyant density properties, and/or positive or negative selection of cell populations using monoclonal antibodies. However, enrichment techniques may significantly change the maturation and/or activation status of DCs or selectively eliminate one or more subpopulations of DCs. To overcome these drawbacks, we designed a multicolor flow cytometric technique for simultaneous analysis of DC populations from tissues of individual mice. The spleens and Peyer's patches were mechanically and enzymatically digested, then incubated with a panel of six monoclonal antibody-fluorochrome direct conjugate reagents. A BD (R) Biosciences LSR II flow cytometer and FCS Express (R) software were used to identify three subtypes of mature DCs (myeloid, lymphoid, and plasmacytoid), precursor DCs, polymorphonuclear neutrophils, B lymphocytes, and Gr-1(+)/CD8 alpha(+) memory T lymphocytes in the spleen. Likewise, we also identified these DC subpopulations and B lymphocytes in the Peyers patches. The three key parameters in analysis of the DC populations were bi-exponential plotting in data analysis, collection of a minimum of 50,000 total events, and accurate color compensation. This procedure to analyze DCs from individual mice can lead to further understanding of the role of DCs in many other model systems as well as better understanding of how dietary or physiological factors may affect in vivo DC homeostasis. (C) 2009 International Society for Advancement of Cytometry