Abstract
Recent advances have revolutionized the oldest high-throughput single-cell analytical tool, flow cytometry. Fluorescent analyzers and sorters with up to seven lasers and the potential to detect up to 50 parameters are changing the way flow cytometry is used, but old school practices which are inadequate for new technologies remain alive. This chapter summarizes recent advances, explains the most salient new features and offers a step-by-step guide to develop and successfully execute high-dimensional fluorescent flow cytometry experiments.
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References
Bagwell CB, Adams EG (1993) Fluorescence spectral overlap compensation for any number of flow cytometry parameters. Ann N Y Acad Sci 677:167–184. https://doi.org/10.1111/j.1749-6632.1993.tb38775.x
Bandura DR, Baranov VI, Ornatsky OI, Antonov A, Kinach R, Lou X, Pavlov S, Vorobiev S, Dick JE, Tanner SD (2009) Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry. Anal Chem 81:6813–6822. https://doi.org/10.1021/ac901049w
Behbehani GK, Bendall SC, Clutter MR, Fantl WJ, Nolan GP (2012) Single-cell mass cytometry adapted to measurements of the cell cycle. Cytometry A 81:552–566. https://doi.org/10.1002/cyto.a.22075
Brummelman J, Haftmann C, Núñez NG, Alvisi G, Mazza EMC, Becher B, Lugli E (2019) Development, application and computational analysis of high-dimensional fluorescent antibody panels for single-cell flow cytometry. Nat Protoc 14:1946–1969. https://doi.org/10.1038/s41596-019-0166-2
Grégori G, Patsekin V, Rajwa B, Jones J, Ragheb K, Holdman C, Robinson JP (2012) Hyperspectral cytometry at the single-cell level using a 32-channel photodetector. Cytometry A 81:35–44. https://doi.org/10.1002/cyto.a.21120
Habbersett RC, Naivar MA, Woods TA, Goddard GR, Graves SW (2007) Evaluation of a green laser pointer for flow cytometry. Cytometry A 71:809–817. https://doi.org/10.1002/cyto.a.20454
Lansdorp PM, Smith C, Safford M, Terstappen LW, Thomas TE (1991) Single laser three color immunofluorescence staining procedures based on energy transfer between phycoerythrin and cyanine 5. Cytometry 12:723–730. https://doi.org/10.1002/cyto.990120806
Li W, Vacca G, Castillo M, Houston KD, Houston JP (2014) Fluorescence lifetime excitation cytometry by kinetic dithering. Electrophoresis 35:1846–1854. https://doi.org/10.1002/elps.201300618
Liechti T, Weber LM, Ashhurst TM, Stanley N, Prlic M, Van Gassen S, Mair F (2021) An updated guide for the perplexed: cytometry in the high-dimensional era. Nat Immunol 22:1190–1197. https://doi.org/10.1038/s41590-021-01006-z
Maecker HT, Trotter J (2006) Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69:1037–1042. https://doi.org/10.1002/cyto.a.20333
Mair F, Tyznik AJ (2019) High-dimensional Immunophenotyping with fluorescence-based cytometry: a practical guidebook. Methods Mol Biol 2032:1–29. https://doi.org/10.1007/978-1-4939-9650-6_1
Barteneva NS, Vorobjev IA (2016) Imaging flow cytometry, 1st edn. Humana Press. https://doi.org/10.1007/978-1-4939-3302-0
Nguyen R, Perfetto S, Mahnke YD, Chattopadhyay P, Roederer M (2013) Quantifying spillover spreading for comparing instrument performance and aiding in multicolor panel design. Cytometry A 83:306–315. https://doi.org/10.1002/cyto.a.22251
Park LM, Lannigan J, Jaimes MC (2020) OMIP-069: forty-color full spectrum flow cytometry panel for deep immunophenotyping of major cell subsets in human peripheral blood. Cytometry A 97:1044–1051. https://doi.org/10.1002/cyto.a.24213
Roederer M, Murphy RF (1986) Cell-by-cell autofluorescence correction for low signal-to-noise systems: application to epidermal growth factor endocytosis by 3T3 fibroblasts. Cytometry 7:558–565. https://doi.org/10.1002/cyto.990070610
Schwann T (1839) Mikroskopische Untersuchungen ueber die Uebereinstimmung in der Struktur und dem Wachstum der Thiere und Pflanzen (Verlag van Sanderschen Buchhandlung)
Wade CG, Rhyne RH Jr, Woodruff WH, Bloch DP, Bartholomew JC (1979) Spectra of cells in flow cytometry using a vidicon detector. J Histochem Cytochem 27:1049–1052. https://doi.org/10.1177/27.6.110874
Acknowledgments
The authors wish to acknowledge Karen Wolcott (CCR/LGI Flow Core) for help with training on the Sony ID7000 spectral analyzer, Jeff Clapper (Sony Biotechnology) for helpful discussions and insight in spectral flow cytometry and Hidehiro Yamane, and Mariah Lee (CCR/LCMB, NCI, NIH) for providing unpublished data in Figs. 6 and 7 for the comparison of conventional and spectral flow cytometry analyses.
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Siddiqui, S., Livák, F. (2023). Principles of Advanced Flow Cytometry: A Practical Guide. In: Bosselut, R., Vacchio, M.S. (eds) T-Cell Development. Methods in Molecular Biology, vol 2580. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2740-2_5
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DOI: https://doi.org/10.1007/978-1-0716-2740-2_5
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