Optimizing a Multicolor Immunophenotyping Assay

ImmunoTechnology Section, Vaccine Research Center, NIAID, NIH, 40 Convent Drive, Room 5509, Bethesda, MD 20892, USA.
Clinics in Laboratory Medicine (Impact Factor: 1.37). 10/2007; 27(3):469-85, v. DOI: 10.1016/j.cll.2007.05.002
Source: PubMed


Flow cytometry-based immunophenotyping assays have become increasingly multiparametric, concomitantly analyzing multiple cellular parameters. To maximize the quality of the information obtained, antibody conjugate panels need to be developed with care, including requisite controls at every step. Such an optimization procedure for multicolor immunophenotyping assays is time consuming, but the value of having a reliable antibody conjugate panel that provides for sensitive detection of all molecules of interest justifies this time investment. This article outlines important considerations and procedures to undertake for the successful design and development of multicolor flow cytometry panels.

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Available from: Yolanda D Mahnke, Dec 01, 2014
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    • "This has been described in detail previously (Wood, 2006; Mahnke & Roederer, 2007). The following checks should be performed: ● Steric hindrance test: Label cells with (i) the complete set of antibodies and (ii) with each individual antibody as single stains. "

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    • "In the last decade, advances in FCM instrumentation and reagent technologies have enabled simultaneous single cell measurement of surface and intracellular markers, including cellular-activation markers, intra-cellular cytokines, immunological signaling, and cytoplasmic and nuclear cell cycle and transcription factors, thus positioning FCM to play an even bigger role in health care and medical research [1-3]. Today’s flow cytometers can measure up to 20 parameters simultaneously – two physical parameters (cell size and granularity) and 18 fluorescent markers [4]. However, the rapid development of FCM instrumentation has outpaced the development of adequate approaches and tools for data analysis. "
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    • "were added to ascertain the previously identified circulating progenitor cell populations in human PB (Estes et al., 2010a,b). All antibodies were titered based on the mean fluorescence intensity with fluorochrome conjugate coupling to specific antigens optimized for the four-antibody plus viability marker panel (Baumgarth and Roederer, 2000; Mahnke and Roederer, 2007; Tung et al., 2007; Estes et al., 2010a). "
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