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Updated: Jun 16, 2026
Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry
Published on: March 5, 2019
Multiplexed immunophenotyping of human antigen-presenting cells in whole blood by polychromatic flow cytometry
Erik Fung1, Laura Esposito, John A Todd
1Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK.
Insights
This study presents two rapid protocols for analyzing human antigen-presenting cells (APCs) in whole blood using flow cytometry. These methods allow for multicolor analysis of APC surface markers in under five hours.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Antigen-presenting cells (APCs) are crucial for initiating adaptive immune responses.
- Characterizing APC subsets and their functional markers in whole blood is essential for immunological studies.
- Existing methods for APC analysis can be time-consuming and require extensive sample manipulation.
Purpose of the Study:
- To develop and validate modular protocols for immunostaining and multiparameter flow cytometric analysis of human APCs.
- To enable simultaneous detection of up to eight colors for comprehensive APC subset characterization.
- To quantify surface expression of key molecules involved in APC function.
Main Methods:
- Developed two modular protocols for immunostaining of whole blood samples.
- Utilized multiparameter flow cytometry with careful selection of monoclonal antibodies (anchor markers).
- Optimized fluorochrome combinations for simultaneous detection of up to eight colors.
Main Results:
- Successfully quantified surface expression of chemotaxis, adhesion, antigen presentation, and immune regulation molecules on circulating APCs.
- Protocols require minimal sample volume (50-100 microliters) and no density-gradient separation.
- The entire procedure, from reagent preparation to flow cytometry, is completed in under 5 hours.
Conclusions:
- The described protocols provide a rapid, efficient, and modular approach for analyzing major human APC subsets in whole blood.
- These methods facilitate detailed characterization of APCs and their functional molecules.
- The protocols are suitable for diverse immunological research applications requiring timely APC analysis.
Abstract:
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.
