A Barcoding Assay for Phenotypic and Functional Characterization of Immune Cells
JoVE Encyclopedia of Experiments
Immunology
0 views • 5:42 min • July 8th, 2025
Begin with test samples containing stimulated and fixed immune cells.
Stimulation induces the expression of specific markers in cells, whereas fixation preserves phenotypic markers, including cell surface antigens, and functional state markers, including cytokines.
Add a unique combination of heavy metal isotope-conjugated antibodies to each sample.
This technique uniquely barcodes multiple samples, distinguishing each sample by its combination of heavy metal isotopes.
The antibodies bind to a common epitope, barcoding all the cells in the samples.
Combine all the barcoded samples in a single tube, for downstream processing.
Add a metal-conjugated antibody cocktail to stain surface antigens.
Add permeabilization buffer to increase cell membrane permeability for intracellular staining.
Add another set of metal-conjugated antibodies to stain intracellular cytokines.
Barcoding enables simultaneous staining and acquisition of the pooled samples via mass cytometry. This technique maximizes assay efficiency, improving the evaluation of phenotypic and functional state markers.
To barcode the lysed fixed cells, thaw the samples from the minus 80 degree Celsius storage, slowly on ice. Dilu
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