Loading…

Appraising the suitability of succinimidyl and lipophilic fluorescent dyes to track proliferation in non-quiescent cells by dye dilution

•Succinimidyl ester dyes generally label cells more efficiently than lipophilic dyes for the purpose of division tracking.•The quality of peak resolution in culture does not always correspond to the fluorescent width of the labelled input population.•This discrepancy correlates with sources of cultu...

Full description

Saved in:
Bibliographic Details
Published in:Methods (San Diego, Calif.) Calif.), 2015-07, Vol.82, p.29-37
Main Authors: Filby, Andrew, Begum, Julfa, Jalal, Marwa, Day, William
Format: Article
Language:English
Subjects:
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
cited_by cdi_FETCH-LOGICAL-c359t-1b3f258c18f71b44c1c677d859ab4ed385f379627b9e3e3612cd2593d2e83ebe3
cites cdi_FETCH-LOGICAL-c359t-1b3f258c18f71b44c1c677d859ab4ed385f379627b9e3e3612cd2593d2e83ebe3
container_end_page 37
container_issue
container_start_page 29
container_title Methods (San Diego, Calif.)
container_volume 82
creator Filby, Andrew
Begum, Julfa
Jalal, Marwa
Day, William
description •Succinimidyl ester dyes generally label cells more efficiently than lipophilic dyes for the purpose of division tracking.•The quality of peak resolution in culture does not always correspond to the fluorescent width of the labelled input population.•This discrepancy correlates with sources of culture-dependent error including dye transfer and daughter cell inheritance. Successful completion of the cell cycle usually results in two identical daughter progeny. This process of generational doubling is termed proliferation and when it occurs in a regulated fashion the benefits range from driving embryonic development to mounting a successful immune response. However when it occurs in a dis-regulated fashion, it is one of the hallmarks of cancer and autoimmunity. These very reasons make proliferation a highly informative parameter in many different biological systems. Conventional flow cytometry (CFC) is a high-throughput, fluorescence-based method for measuring the phenotype and function of cells. The application of CFC to measuring proliferation requires a fluorescent dye able to mark live cells so that when they divide, the daughter progeny receives approximately half the fluorescence of the parent. In measurement space, this translates into peaks of fluorescence decreasing by approximately half, each corresponding to a round of division. It is essential that these peaks can be resolved from one another otherwise it is nearly impossible to obtain accurate quantitative proliferation data. Peak resolution is affected by many things, including instrument performance, the choice of fluorescent dye and the inherent properties of the cells under investigation. There are now many fluorescent dyes available for tracking proliferation by dye dilution differing in their chemistry and spectral properties. Here we provide a method for assessing the performance of various candidate dyes with particular emphasis on situations where the cell type is non-quiescent. We have shown previously that even under optimised instrument and labelling conditions, the heterogeneity of non-quiescent cells makes it impossible to obtain an input width below the threshold for peak resolution without reducing the fluorescence distribution using a cell sorter. Moreover, our method also measures how the dye performs post-labelling in terms of loss/transfer to other cells and how the dye is inherited across the cytokinetic plane. All of these factors will affect peak resolution both in non-qui
doi_str_mv 10.1016/j.ymeth.2015.02.016
format article
fullrecord <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_1689310718</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><els_id>S1046202315000894</els_id><sourcerecordid>1689310718</sourcerecordid><originalsourceid>FETCH-LOGICAL-c359t-1b3f258c18f71b44c1c677d859ab4ed385f379627b9e3e3612cd2593d2e83ebe3</originalsourceid><addsrcrecordid>eNp9kcuOFSEQhonROOPoE5gYlm665XL6tnAxmYyXZBI3uiY0VHvqSEMP0Cb9Bj62tOfo0hVQfH_VDz8hrzmrOePtu1O9zZCPtWC8qZmoS-0JueZsaKqBS_Z03x_aSjAhr8iLlE6MMS66_jm5Ek3PBOftNfl1uyxRY0L_neYj0LRi1iM6zBsNUzkagx5ntJuj2lvqcAnLsdwbOrk1REgGfKZ2g0RzoDlq84MuMTicIOqMwVP01AdfPa54gQ04l-i47Spq0a079pI8m7RL8Oqy3pBvH-6_3n2qHr58_Hx3-1AZ2Qy54qOcinvD-6nj4-FguGm7zvbNoMcDWNk3k-yGVnTjABJky4WxohmkFdBLGEHekLfnvsXk4wopqxnT7kh7CGtSvO0HyVnH-4LKM2piSCnCpJaIs46b4kztEaiT-hOB2iNQTKhSK6o3lwHrOIP9p_n75wV4fwagPPMnQlTJIHgDFiOYrGzA_w74DbLInBk</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>1689310718</pqid></control><display><type>article</type><title>Appraising the suitability of succinimidyl and lipophilic fluorescent dyes to track proliferation in non-quiescent cells by dye dilution</title><source>Elsevier:Jisc Collections:Elsevier Read and Publish Agreement 2022-2024:Freedom Collection (Reading list)</source><creator>Filby, Andrew ; Begum, Julfa ; Jalal, Marwa ; Day, William</creator><creatorcontrib>Filby, Andrew ; Begum, Julfa ; Jalal, Marwa ; Day, William</creatorcontrib><description>•Succinimidyl ester dyes generally label cells more efficiently than lipophilic dyes for the purpose of division tracking.•The quality of peak resolution in culture does not always correspond to the fluorescent width of the labelled input population.•This discrepancy correlates with sources of culture-dependent error including dye transfer and daughter cell inheritance. Successful completion of the cell cycle usually results in two identical daughter progeny. This process of generational doubling is termed proliferation and when it occurs in a regulated fashion the benefits range from driving embryonic development to mounting a successful immune response. However when it occurs in a dis-regulated fashion, it is one of the hallmarks of cancer and autoimmunity. These very reasons make proliferation a highly informative parameter in many different biological systems. Conventional flow cytometry (CFC) is a high-throughput, fluorescence-based method for measuring the phenotype and function of cells. The application of CFC to measuring proliferation requires a fluorescent dye able to mark live cells so that when they divide, the daughter progeny receives approximately half the fluorescence of the parent. In measurement space, this translates into peaks of fluorescence decreasing by approximately half, each corresponding to a round of division. It is essential that these peaks can be resolved from one another otherwise it is nearly impossible to obtain accurate quantitative proliferation data. Peak resolution is affected by many things, including instrument performance, the choice of fluorescent dye and the inherent properties of the cells under investigation. There are now many fluorescent dyes available for tracking proliferation by dye dilution differing in their chemistry and spectral properties. Here we provide a method for assessing the performance of various candidate dyes with particular emphasis on situations where the cell type is non-quiescent. We have shown previously that even under optimised instrument and labelling conditions, the heterogeneity of non-quiescent cells makes it impossible to obtain an input width below the threshold for peak resolution without reducing the fluorescence distribution using a cell sorter. Moreover, our method also measures how the dye performs post-labelling in terms of loss/transfer to other cells and how the dye is inherited across the cytokinetic plane. All of these factors will affect peak resolution both in non-quiescent and primary cell types.</description><identifier>ISSN: 1046-2023</identifier><identifier>EISSN: 1095-9130</identifier><identifier>DOI: 10.1016/j.ymeth.2015.02.016</identifier><identifier>PMID: 25802116</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Cell Proliferation - physiology ; Dye dilution ; Flow Cytometry ; Fluorescent Dyes ; Humans ; Imaging flow cytometry ; Proliferation tracking ; Succinimides</subject><ispartof>Methods (San Diego, Calif.), 2015-07, Vol.82, p.29-37</ispartof><rights>2015 Elsevier Inc.</rights><rights>Copyright © 2015 Elsevier Inc. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c359t-1b3f258c18f71b44c1c677d859ab4ed385f379627b9e3e3612cd2593d2e83ebe3</citedby><cites>FETCH-LOGICAL-c359t-1b3f258c18f71b44c1c677d859ab4ed385f379627b9e3e3612cd2593d2e83ebe3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25802116$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Filby, Andrew</creatorcontrib><creatorcontrib>Begum, Julfa</creatorcontrib><creatorcontrib>Jalal, Marwa</creatorcontrib><creatorcontrib>Day, William</creatorcontrib><title>Appraising the suitability of succinimidyl and lipophilic fluorescent dyes to track proliferation in non-quiescent cells by dye dilution</title><title>Methods (San Diego, Calif.)</title><addtitle>Methods</addtitle><description>•Succinimidyl ester dyes generally label cells more efficiently than lipophilic dyes for the purpose of division tracking.•The quality of peak resolution in culture does not always correspond to the fluorescent width of the labelled input population.•This discrepancy correlates with sources of culture-dependent error including dye transfer and daughter cell inheritance. Successful completion of the cell cycle usually results in two identical daughter progeny. This process of generational doubling is termed proliferation and when it occurs in a regulated fashion the benefits range from driving embryonic development to mounting a successful immune response. However when it occurs in a dis-regulated fashion, it is one of the hallmarks of cancer and autoimmunity. These very reasons make proliferation a highly informative parameter in many different biological systems. Conventional flow cytometry (CFC) is a high-throughput, fluorescence-based method for measuring the phenotype and function of cells. The application of CFC to measuring proliferation requires a fluorescent dye able to mark live cells so that when they divide, the daughter progeny receives approximately half the fluorescence of the parent. In measurement space, this translates into peaks of fluorescence decreasing by approximately half, each corresponding to a round of division. It is essential that these peaks can be resolved from one another otherwise it is nearly impossible to obtain accurate quantitative proliferation data. Peak resolution is affected by many things, including instrument performance, the choice of fluorescent dye and the inherent properties of the cells under investigation. There are now many fluorescent dyes available for tracking proliferation by dye dilution differing in their chemistry and spectral properties. Here we provide a method for assessing the performance of various candidate dyes with particular emphasis on situations where the cell type is non-quiescent. We have shown previously that even under optimised instrument and labelling conditions, the heterogeneity of non-quiescent cells makes it impossible to obtain an input width below the threshold for peak resolution without reducing the fluorescence distribution using a cell sorter. Moreover, our method also measures how the dye performs post-labelling in terms of loss/transfer to other cells and how the dye is inherited across the cytokinetic plane. All of these factors will affect peak resolution both in non-quiescent and primary cell types.</description><subject>Cell Proliferation - physiology</subject><subject>Dye dilution</subject><subject>Flow Cytometry</subject><subject>Fluorescent Dyes</subject><subject>Humans</subject><subject>Imaging flow cytometry</subject><subject>Proliferation tracking</subject><subject>Succinimides</subject><issn>1046-2023</issn><issn>1095-9130</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><recordid>eNp9kcuOFSEQhonROOPoE5gYlm665XL6tnAxmYyXZBI3uiY0VHvqSEMP0Cb9Bj62tOfo0hVQfH_VDz8hrzmrOePtu1O9zZCPtWC8qZmoS-0JueZsaKqBS_Z03x_aSjAhr8iLlE6MMS66_jm5Ek3PBOftNfl1uyxRY0L_neYj0LRi1iM6zBsNUzkagx5ntJuj2lvqcAnLsdwbOrk1REgGfKZ2g0RzoDlq84MuMTicIOqMwVP01AdfPa54gQ04l-i47Spq0a079pI8m7RL8Oqy3pBvH-6_3n2qHr58_Hx3-1AZ2Qy54qOcinvD-6nj4-FguGm7zvbNoMcDWNk3k-yGVnTjABJky4WxohmkFdBLGEHekLfnvsXk4wopqxnT7kh7CGtSvO0HyVnH-4LKM2piSCnCpJaIs46b4kztEaiT-hOB2iNQTKhSK6o3lwHrOIP9p_n75wV4fwagPPMnQlTJIHgDFiOYrGzA_w74DbLInBk</recordid><startdate>20150701</startdate><enddate>20150701</enddate><creator>Filby, Andrew</creator><creator>Begum, Julfa</creator><creator>Jalal, Marwa</creator><creator>Day, William</creator><general>Elsevier Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20150701</creationdate><title>Appraising the suitability of succinimidyl and lipophilic fluorescent dyes to track proliferation in non-quiescent cells by dye dilution</title><author>Filby, Andrew ; Begum, Julfa ; Jalal, Marwa ; Day, William</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c359t-1b3f258c18f71b44c1c677d859ab4ed385f379627b9e3e3612cd2593d2e83ebe3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Cell Proliferation - physiology</topic><topic>Dye dilution</topic><topic>Flow Cytometry</topic><topic>Fluorescent Dyes</topic><topic>Humans</topic><topic>Imaging flow cytometry</topic><topic>Proliferation tracking</topic><topic>Succinimides</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Filby, Andrew</creatorcontrib><creatorcontrib>Begum, Julfa</creatorcontrib><creatorcontrib>Jalal, Marwa</creatorcontrib><creatorcontrib>Day, William</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Methods (San Diego, Calif.)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Filby, Andrew</au><au>Begum, Julfa</au><au>Jalal, Marwa</au><au>Day, William</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Appraising the suitability of succinimidyl and lipophilic fluorescent dyes to track proliferation in non-quiescent cells by dye dilution</atitle><jtitle>Methods (San Diego, Calif.)</jtitle><addtitle>Methods</addtitle><date>2015-07-01</date><risdate>2015</risdate><volume>82</volume><spage>29</spage><epage>37</epage><pages>29-37</pages><issn>1046-2023</issn><eissn>1095-9130</eissn><abstract>•Succinimidyl ester dyes generally label cells more efficiently than lipophilic dyes for the purpose of division tracking.•The quality of peak resolution in culture does not always correspond to the fluorescent width of the labelled input population.•This discrepancy correlates with sources of culture-dependent error including dye transfer and daughter cell inheritance. Successful completion of the cell cycle usually results in two identical daughter progeny. This process of generational doubling is termed proliferation and when it occurs in a regulated fashion the benefits range from driving embryonic development to mounting a successful immune response. However when it occurs in a dis-regulated fashion, it is one of the hallmarks of cancer and autoimmunity. These very reasons make proliferation a highly informative parameter in many different biological systems. Conventional flow cytometry (CFC) is a high-throughput, fluorescence-based method for measuring the phenotype and function of cells. The application of CFC to measuring proliferation requires a fluorescent dye able to mark live cells so that when they divide, the daughter progeny receives approximately half the fluorescence of the parent. In measurement space, this translates into peaks of fluorescence decreasing by approximately half, each corresponding to a round of division. It is essential that these peaks can be resolved from one another otherwise it is nearly impossible to obtain accurate quantitative proliferation data. Peak resolution is affected by many things, including instrument performance, the choice of fluorescent dye and the inherent properties of the cells under investigation. There are now many fluorescent dyes available for tracking proliferation by dye dilution differing in their chemistry and spectral properties. Here we provide a method for assessing the performance of various candidate dyes with particular emphasis on situations where the cell type is non-quiescent. We have shown previously that even under optimised instrument and labelling conditions, the heterogeneity of non-quiescent cells makes it impossible to obtain an input width below the threshold for peak resolution without reducing the fluorescence distribution using a cell sorter. Moreover, our method also measures how the dye performs post-labelling in terms of loss/transfer to other cells and how the dye is inherited across the cytokinetic plane. All of these factors will affect peak resolution both in non-quiescent and primary cell types.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>25802116</pmid><doi>10.1016/j.ymeth.2015.02.016</doi><tpages>9</tpages></addata></record>
fulltext fulltext
identifier ISSN: 1046-2023
ispartof Methods (San Diego, Calif.), 2015-07, Vol.82, p.29-37
issn 1046-2023
1095-9130
language eng
recordid cdi_proquest_miscellaneous_1689310718
source Elsevier:Jisc Collections:Elsevier Read and Publish Agreement 2022-2024:Freedom Collection (Reading list)
subjects Cell Proliferation - physiology
Dye dilution
Flow Cytometry
Fluorescent Dyes
Humans
Imaging flow cytometry
Proliferation tracking
Succinimides
title Appraising the suitability of succinimidyl and lipophilic fluorescent dyes to track proliferation in non-quiescent cells by dye dilution
url http://sfxeu10.hosted.exlibrisgroup.com/loughborough?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-25T00%3A27%3A59IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_cross&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Appraising%20the%20suitability%20of%20succinimidyl%20and%20lipophilic%20fluorescent%20dyes%20to%20track%20proliferation%20in%20non-quiescent%20cells%20by%20dye%20dilution&rft.jtitle=Methods%20(San%20Diego,%20Calif.)&rft.au=Filby,%20Andrew&rft.date=2015-07-01&rft.volume=82&rft.spage=29&rft.epage=37&rft.pages=29-37&rft.issn=1046-2023&rft.eissn=1095-9130&rft_id=info:doi/10.1016/j.ymeth.2015.02.016&rft_dat=%3Cproquest_cross%3E1689310718%3C/proquest_cross%3E%3Cgrp_id%3Ecdi_FETCH-LOGICAL-c359t-1b3f258c18f71b44c1c677d859ab4ed385f379627b9e3e3612cd2593d2e83ebe3%3C/grp_id%3E%3Coa%3E%3C/oa%3E%3Curl%3E%3C/url%3E&rft_id=info:oai/&rft_pqid=1689310718&rft_id=info:pmid/25802116&rfr_iscdi=true