Immunophenotyping using flow cytometry highly benefits from multiplexing samples for generation of more robust data, because of reduction of antibody consumption, batch effect and technical variations. One way to multiplex is via fluorescent cell barcoding (FCB) prior to staining procedure.
FCB is a high-throughput multiplexed assay using various concentrations of different fluorescent dyes. Individual samples are uniquely labeled, then mixed together, stained and analyzed as a single sample, decreasing technical variations and increasing throughput and speed of acquisition. In addition, FCB simplifies implementation of normalization using a bridge control sample.
In this chapter, we illustrate the protocol for FCB and recommendations for choosing barcoding dyes and concentrations among other technical considerations.
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