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Updated: May 20, 2026
Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo
Published on: February 5, 2021
PE-Cy5.5 conjugates bind to the cells expressing mouse DEC205/CD205.
Chae Gyu Park1, Anthony Rodriguez, Ralph M Steinman
1Laboratory of Cellular Physiology and Immunology and Chris Browne Center for Immunology and Immune Diseases, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. parkc@rockefeller.edu
Researchers found that PE-Cy5.5 conjugates bind non-specifically to mouse DEC205 (mDEC205)-expressing cells. This unexpected interaction requires caution when using PE-Cy5.5 conjugates in multicolor flow cytometry with mDEC205-positive cells.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- DEC205/CD205 is a C-type multilectin endocytic receptor highly expressed on dendritic cells (DCs).
- DEC205 facilitates antigen delivery to DC machinery, leading to DC-targeted vaccines using anti-DC monoclonal antibodies (mAbs).
- Flow cytometry is crucial for characterizing anti-DC mAbs.
Purpose of the Study:
- To investigate the unexpected strong binding of secondary antibodies to mouse DEC205 (mDEC205)-expressing cells during flow cytometric analysis.
- To characterize the specificity of this non-specific binding phenomenon with various conjugates and cell types.
- To provide guidance on the use of PE-Cy5.5 conjugates in flow cytometry involving mDEC205.
Main Methods:
- Flow cytometric analysis of cells expressing mouse DEC205 (mDEC205) and human DEC205.
- Incubation of cells with various secondary antibodies and streptavidin conjugates, including PE-Cy5.5, PerCP-Cy5.5, APC-Cy5.5, Cy5.5, PE, PE-Cy5, Cy5, FITC, and Alexa488.
- Testing binding on mDEC205-expressing cell lines (CHO, KIT6, HEK293) and human DEC205-expressing cells.
Main Results:
- Secondary anti-immunoglobulin antibodies conjugated with PE-Cy5.5 bound strongly to mDEC205-expressing cells without primary anti-mDEC205 mAb.
- Various PE-Cy5.5 conjugates (antibodies and streptavidin) showed binding to mDEC205-expressing cells, but not to human DEC205-expressing cells.
- Only PE-Cy5.5 conjugates exhibited strong binding; other conjugates (PerCP-Cy5.5, APC-Cy5.5, Cy5.5) bound weakly, while PE, PE-Cy5, Cy5, FITC, or Alexa488 conjugates did not bind.
Conclusions:
- PE-Cy5.5 conjugates exhibit non-specific binding to cells expressing mouse DEC205 (mDEC205).
- This binding is specific to mDEC205 and the PE-Cy5.5 fluorochrome, distinguishing it from other conjugates.
- Caution is advised when using PE-Cy5.5 conjugates in multicolor flow cytometry protocols involving mDEC205-positive cells to avoid misinterpretation of results.
