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. 2018:1678:139-150.
doi: 10.1007/978-1-4939-7346-0_8.

Multiparameter Conventional Flow Cytometry

Katherine M McKinnon  1
Affiliations

Multiparameter Conventional Flow Cytometry

Katherine M McKinnon. Methods Mol Biol. 2018.

Abstract

Multicolor flow cytometry is a useful technique when examining mixed populations of cells, such as blood and tissue cells in human and animal samples. The ability to use multiple fluorescent markers simultaneously allows for the identification of multiple cell types, as well as functional markers that further characterize each sample. The introduction of instruments capable of measuring 12-plus colors and new reagents has made this type of flow cytometry both popular and problematic. Adapting a typical staining panel from 4 to 6 color tubes to more than 12 colors is not simply a matter of "plug and play", but must be approached in a systematic manner to achieve a successful multi-parameter staining panel. This chapter will examine the considerations and methods needed to successfully perform multicolor flow cytometry.

Keywords: Flow cytometry; Immunology; Multi-parameter; Multicolor flow cytometry; Staining.

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Figures

Fig. 1
Fig. 1
Instrument configuration for LSRII (BD Biosciences) and FACSymphony (BD Biosciences) flow cytometers in the NCI Vaccine Branch Flow Cytometry Core Facility. The tables include dichroic and bandpass filters for each detector. Recommended dyes are also given for each detector from multiple manufacturers. When designing a staining panel, one fluorochrome should be chosen for each detector
Fig. 2
Fig. 2
Example of antibody titration using Stain Index (SI). Non-human primate PBMC were stained with different volumes of CD4 PerCP-Cy5.5 (clone L200, BD Biosciences) and then acquired using a LSRII flow cytometer. Data was analyzed in FlowJo 10.2 software (FlowJo, LLC, Ashland, OR) and the stain index was calculated using the formula SI = D/W
Fig. 3
Fig. 3
Example of compensation setup using FACSDiva 8 software. COMPTrol beads were stained with PerCP-Cy5.5 and then examined on a LSRII flow cytometer for spill-over into other fluorescent channels. Since the fluorescence signal was strongest in the PerCP-Cy5.5 channel, the detector settings were considered correct
Fig. 4
Fig. 4
Sample staining panels for LSRII and FACSymphony
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