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We describe two modular protocols for immunostaining and multiparameter flow cytometric analysis of major human antigen-presenting cells (APCs; e.g., dendritic cells, monocytes and B lymphocytes) in minimally manipulated whole blood samples. Simultaneous detection of up to eight colors is enabled by careful selection and testing of cell-subset-defining monoclonal antibodies (anchor markers) in the appropriate fluorochrome combinations, in order to show the quantification of surface expression levels of molecules involved in chemotaxis (e.g., CX(3)CR1 and CCR2), adhesion (e.g., CD11b and CD62L), antigen presentation (e.g., CD83, CD86 and CD209) and immune regulation (e.g., CD101) on circulating APCs. Each immunostaining reaction requires as little as 50-100 microl of peripheral whole blood and no density-gradient separation, and the entire procedure from preparation of reagents to flow cytometry can be completed in <5 h.

Original publication

DOI

10.1038/nprot.2009.246

Type

Journal article

Journal

Nat Protoc

Publication Date

02/2010

Volume

5

Pages

357 - 370

Keywords

Antibodies, Antigen-Presenting Cells, Antigens, CD, Antigens, CD11, B-Lymphocytes, Dendritic Cells, Erythrocytes, Flow Cytometry, HLA-DR Antigens, Humans, Immunoglobulin Isotypes, Immunophenotyping, Monocytes, Receptors, CCR2, Receptors, Interleukin-8A