Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells
| Autor: | Stephanie N. Johnson, Kyle D. Apley, J. Daniel Griffin, Brandon J. DeKosky, Cory Berkland |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Fluorophore
Multiple Sclerosis General Chemical Engineering B-cell receptor 02 engineering and technology General Biochemistry Genetics and Molecular Biology Flow cytometry Polymerization 03 medical and health sciences chemistry.chemical_compound Mice Antigen medicine Animals Humans Antigens B cell 030304 developmental biology Fluorescent Dyes CD86 0303 health sciences B-Lymphocytes Bioconjugation General Immunology and Microbiology medicine.diagnostic_test General Neuroscience 021001 nanoscience & nanotechnology Fluorescence Cell biology medicine.anatomical_structure Diabetes Mellitus Type 1 chemistry Click Chemistry 0210 nano-technology |
| Zdroj: | Journal of visualized experiments : JoVE. (164) |
| ISSN: | 1940-087X |
| Popis: | Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and the use of four-arm, fluorescent PEG-antigen conjugates to selectively identify antigen-specific B cells through avid engagement with cognate B cell receptors. Using modular click chemistry and commercially available fluorophore kit chemistries, we demonstrated the versatility of preparing customized fluorescent PEG-conjugates by creating distinct arrays for proteolipid protein (PLP139-151) and insulin, which are important autoantigens in murine models of multiple sclerosis and type 1 diabetes, respectively. Assays were developed for each fluorescent conjugate in its respective disease model using flow cytometry. Antigen arrays were compared to monovalent autoantigen to quantify the benefit of multimerization onto PEG backbones. Finally, we illustrated the utility of this platform by isolating and assessing anti-insulin B cell responses after antigen stimulation ex vivo. Labeling insulin-specific B cells enabled the amplified detection of changes to co-stimulation (CD86) that were otherwise dampened in aggregate B cell analysis. Together, this report enables the production and use of fluorescent antigen arrays as a robust tool for probing B cell populations. |
| Databáze: | OpenAIRE |
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