Cargando…

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

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Futamura, Koji, Sekino, Masashi, Hata, Akihiro, Ikebuchi, Ryoyo, Nakanishi, Yasutaka, Egawa, Gyohei, Kabashima, Kenji, Watanabe, Takeshi, Furuki, Motohiro, Tomura, Michio
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5132038/
https://www.ncbi.nlm.nih.gov/pubmed/26217952
http://dx.doi.org/10.1002/cyto.a.22725
_version_ 1782470990498889728
author Futamura, Koji
Sekino, Masashi
Hata, Akihiro
Ikebuchi, Ryoyo
Nakanishi, Yasutaka
Egawa, Gyohei
Kabashima, Kenji
Watanabe, Takeshi
Furuki, Motohiro
Tomura, Michio
author_facet Futamura, Koji
Sekino, Masashi
Hata, Akihiro
Ikebuchi, Ryoyo
Nakanishi, Yasutaka
Egawa, Gyohei
Kabashima, Kenji
Watanabe, Takeshi
Furuki, Motohiro
Tomura, Michio
author_sort Futamura, Koji
collection PubMed
description 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. © 2015 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC
format Online
Article
Text
id pubmed-5132038
institution National Center for Biotechnology Information
language English
publishDate 2015
publisher John Wiley and Sons Inc.
record_format MEDLINE/PubMed
spelling pubmed-51320382016-12-02 Novel full‐spectral flow cytometry with multiple spectrally‐adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement Futamura, Koji Sekino, Masashi Hata, Akihiro Ikebuchi, Ryoyo Nakanishi, Yasutaka Egawa, Gyohei Kabashima, Kenji Watanabe, Takeshi Furuki, Motohiro Tomura, Michio Cytometry A Original Articles 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. © 2015 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC John Wiley and Sons Inc. 2015-07-28 2015-09 /pmc/articles/PMC5132038/ /pubmed/26217952 http://dx.doi.org/10.1002/cyto.a.22725 Text en © 2015 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC This is an open access article under the terms of the Creative Commons Attribution (http://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Articles
Futamura, Koji
Sekino, Masashi
Hata, Akihiro
Ikebuchi, Ryoyo
Nakanishi, Yasutaka
Egawa, Gyohei
Kabashima, Kenji
Watanabe, Takeshi
Furuki, Motohiro
Tomura, Michio
Novel full‐spectral flow cytometry with multiple spectrally‐adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement
title Novel full‐spectral flow cytometry with multiple spectrally‐adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement
title_full Novel full‐spectral flow cytometry with multiple spectrally‐adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement
title_fullStr Novel full‐spectral flow cytometry with multiple spectrally‐adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement
title_full_unstemmed Novel full‐spectral flow cytometry with multiple spectrally‐adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement
title_short Novel full‐spectral flow cytometry with multiple spectrally‐adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement
title_sort novel full‐spectral flow cytometry with multiple spectrally‐adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement
topic Original Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5132038/
https://www.ncbi.nlm.nih.gov/pubmed/26217952
http://dx.doi.org/10.1002/cyto.a.22725
work_keys_str_mv AT futamurakoji novelfullspectralflowcytometrywithmultiplespectrallyadjacentfluorescentproteinsandfluorochromesandvisualizationofinvivocellularmovement
AT sekinomasashi novelfullspectralflowcytometrywithmultiplespectrallyadjacentfluorescentproteinsandfluorochromesandvisualizationofinvivocellularmovement
AT hataakihiro novelfullspectralflowcytometrywithmultiplespectrallyadjacentfluorescentproteinsandfluorochromesandvisualizationofinvivocellularmovement
AT ikebuchiryoyo novelfullspectralflowcytometrywithmultiplespectrallyadjacentfluorescentproteinsandfluorochromesandvisualizationofinvivocellularmovement
AT nakanishiyasutaka novelfullspectralflowcytometrywithmultiplespectrallyadjacentfluorescentproteinsandfluorochromesandvisualizationofinvivocellularmovement
AT egawagyohei novelfullspectralflowcytometrywithmultiplespectrallyadjacentfluorescentproteinsandfluorochromesandvisualizationofinvivocellularmovement
AT kabashimakenji novelfullspectralflowcytometrywithmultiplespectrallyadjacentfluorescentproteinsandfluorochromesandvisualizationofinvivocellularmovement
AT watanabetakeshi novelfullspectralflowcytometrywithmultiplespectrallyadjacentfluorescentproteinsandfluorochromesandvisualizationofinvivocellularmovement
AT furukimotohiro novelfullspectralflowcytometrywithmultiplespectrallyadjacentfluorescentproteinsandfluorochromesandvisualizationofinvivocellularmovement
AT tomuramichio novelfullspectralflowcytometrywithmultiplespectrallyadjacentfluorescentproteinsandfluorochromesandvisualizationofinvivocellularmovement