鉴定和计数来自小鼠脾脏和Peyer小结的树突状细胞

niwanmao

标题：用流式细胞术鉴定和计数来自小鼠脾脏和Peyer小结的树突状细胞

Cytometry Part A 

Volume 75A Issue 11, Pages 951 - 959

Published Online: 9 Sep 2009

The identification and enumeration of dendritic cell populations from individual mouse spleen and Peyer's patches using flow cytometric analysis

David M. Duriancik 1, Kathleen A. Hoag 1 2 *

1Department of Food Science and Human Nutrition, Michigan State University, East Lansing, Michigan 48824 2Biomedical Laboratory Diagnostic Program, Michigan State University, East Lansing, Michigan 48824

email: Kathleen A. Hoag ([email]hoagk@msu.edu[/email])

^*Correspondence to Kathleen A. Hoag, Biomedical Laboratory Diagnostics Program, Michigan State University, 322 North Kedzie Hall, East Lansing, MI 48824-1031

 

flow cytometry • dendritic cells • spleen • Peyer's patches • mice

 

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® Biosciences LSR II flow cytometer and FCS Express® software were used to identify three subtypes of mature DCs (myeloid, lymphoid, and plasmacytoid), precursor DCs, polymorphonuclear neutrophils, B lymphocytes, and Gr-1+/CD8a+ memory T lymphocytes in the spleen. Likewise, we also identified these DC subpopulations and B lymphocytes in the Peyer's 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. © 2009 International Society for Advancement of Cytometry

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Received: 22 June 2009; Revised: 12 August 2009; Accepted: 18 August 2009