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-permeabilization 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.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
DesMarais V,Eddy RJ,Sharma VP,Stone O,Condeelis JSdoi
10.2144/btn-2018-0112subject
Has Abstractpub_date
2019-03-01 00:00:00pages
113-119issue
3eissn
0736-6205issn
1940-9818journal_volume
66pub_type
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