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Cytometry A. 2015 Sep;87(9):830-42. doi: 10.1002/cyto.a.22725. Epub 2015 Jul 28.

Novel full-spectral flow cytometry with multiple spectrally-adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement.

Author information

1
FCM Business Department, Life Science Business Division, Medical Business Unit, Sony Corporation, Minato-Ku, Tokyo, 108-0075, Japan.
2
Concept Development Department, Application Technology Development Division, System R&D Group, RDS Platform, Sony Corporation, Shinagawa-Ku, Tokyo, 141-0001, Japan.
3
Center for Innovation in Immunoregulative Technology and Therapeutics, Kyoto University Graduate School of Medicine, Yoshida-Konoe, Kyoto, 606-8501, Japan.
4
Laboratory of Immunology, Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiorikita, Tondabayashi-City, Osaka Prefecture, 584-8540, Japan.
5
Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, 606-8501, Japan.
6
The Tazuke-Kofukai Medical Research Institute/Kitano Hospital, 2-4-20 Ohgimachi, Kita-Ku, Osaka, 530-8480, Japan.

Abstract

Flow cytometric analysis with multicolor fluoroprobes is an essential method for detecting biological signatures of cells. Here, we present a new full-spectral flow cytometer (spectral-FCM). Unlike conventional flow cytometer, this spectral-FCM acquires the emitted fluorescence for all probes across the full-spectrum from each cell with 32 channels sequential PMT unit after dispersion with prism, and extracts the signals of each fluoroprobe based on the spectral shape of each fluoroprobe using unique algorithm in high speed, high sensitive, accurate, automatic and real-time. The spectral-FCM detects the continuous changes in emission spectra from green to red of the photoconvertible protein, KikGR with high-spectral resolution and separates spectrally-adjacent fluoroprobes, such as FITC (Emission peak (Em) 519 nm) and EGFP (Em 507 nm). Moreover, the spectral-FCM can measure and subtract autofluorescence of each cell providing increased signal-to-noise ratios and improved resolution of dim samples, which leads to a transformative technology for investigation of single cell state and function. These advances make it possible to perform 11-color fluorescence analysis to visualize movement of multilinage immune cells by using KikGR-expressing mice. Thus, the novel spectral flow cytometry improves the combinational use of spectrally-adjacent various FPs and multicolor fluorochromes in metabolically active cell for the investigation of not only the immune system but also other research and clinical fields of use.

KEYWORDS:

immune cell movement; photoconvertible fluorescent protein; separation of spectrally-adjacent fluoroprobes; spectral flow cytometry

PMID:
26217952
PMCID:
PMC5132038
DOI:
10.1002/cyto.a.22725
[Indexed for MEDLINE]
Free PMC Article

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