Giudice Valentina, Fantoni Giovanna, Biancotto Angélique
Department of Medicine, Surgery, and Dentistry, Scuola Medica Salernitana, University of Salerno, Baronissi, Italy.
Center for Human Immunology, NIAID, NIH, Bethesda, MD, USA.
Methods Mol Biol. 2019;2032:53-68. doi: 10.1007/978-1-4939-9650-6_3.
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.
使用流式细胞术进行免疫表型分析通过对样本进行多重检测以生成更可靠的数据而受益匪浅，这是因为减少了抗体消耗、批次效应和技术差异。一种多重检测的方法是在染色程序之前通过荧光细胞条形码（FCB）实现。FCB是一种使用不同浓度的各种荧光染料的高通量多重检测方法。单个样本被独特标记，然后混合在一起，作为单个样本进行染色和分析，减少了技术差异，提高了通量和采集速度。此外，FCB简化了使用桥接对照样本进行标准化的实施过程。在本章中，我们阐述了FCB的方案以及在其他技术考虑因素中选择条形码染料和浓度的建议。
