@article{97e4ec7f7251411aa5b6fe91aa00f6cc,
title = "Optimizing leading edge F-actin labeling using multiple actin probes, fixation methods and imaging modalities",
abstract = "We systematically evaluated the performance and reliability of several widely used, commercially available actin-filament probes in a highly motile breast adenocarcinoma cell line to optimize the visualization of F-actin-rich dynamic lamellipodia. We evaluated four Phalloidin-fluorophores, two anti-actin antibodies, and three live-cell actin probes in five fixation conditions across three imaging platforms as a basis for the design of optimized protocols. Of the fluorescent phalloidin-dye conjugates tested, Alexa Fluor-488 Phalloidin ranked best in overall labeling of the actin cytoskeleton and maintenance of the fluorescence signal over time. Use of actin monoclonal antibodies revealed significant limitations under a variety of fixation-permeabili-zation conditions. Evaluation of commonly used live-cell probes provides evidence for actin filament bias, with TagRFP-Lifeact excluded from lamellipodia, but not mEGFP-Lifeact or F-tractin-EGFP.",
keywords = "Actin, Cell motility, F-tractin, Lamellipodia, Lifeact, Phalloidin",
author = "Vera DesMarais and Eddy, {Robert J.} and Sharma, {Ved P.} and Orrin Stone and Condeelis, {John S.}",
note = "Funding Information: All imaging was conducted in the Analytical Imaging Facility (AIF) (funded by NCI Cancer Grant P30CA013330). SIM imaging was executed on the Nikon-STORM/SIM/ TIRF microscope (funded by NIH 1S10OD18218-1). Some confocal imaging was executed on the Leica SP8 confocal microscope (funded by NIH 1S10OD023591-01). The research was funded by NIH grants CA150344 and CA216248 (RE, VS, JC). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. Funding Information: All?imaging?was?conducted?in?the?Analytical? Imaging?Facility?(AIF)?(funded?by?NCI? Cancer? Grant? P30CA013330).? SIM? imaging? was?executed?on?the?Nikon-STORM/SIM/ TIRF?microscope?(funded?by?NIH? 1S10OD18218–1).? Some? confocal? imaging? was?executed?on?the?Leica?SP8?confocal? microscope?(funded?by?NIH? 1S10OD023591–01).?The?research?was? funded?by?NIH?grants?CA150344?and? CA216248?(RE,?VS,?JC).?The?authors?have? no?other?relevant?affiliations?or?financial? involvement? with? any? organization? or? entity? with?a?financial?interest?in?or?financial? conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. Publisher Copyright: {\textcopyright} 2019 John S Condeelis",
year = "2019",
month = mar,
doi = "10.2144/btn-2018-0112",
language = "English (US)",
volume = "66",
pages = "113--119",
journal = "BioTechniques",
issn = "0736-6205",
publisher = "Eaton Publishing Company",
number = "3",
}