Optimizing leading edge F-actin labeling using multiple actin probes, fixation methods and imaging modalities

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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.

Original languageEnglish (US)
Pages (from-to)113-119
Number of pages7
JournalBioTechniques
Volume66
Issue number3
DOIs
StatePublished - Mar 1 2019

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Keywords

  • Actin
  • Cell motility
  • F-tractin
  • Lamellipodia
  • Lifeact
  • Phalloidin

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

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