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-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

Biotechniques

journal_title

BioTechniques

authors

DesMarais V,Eddy RJ,Sharma VP,Stone O,Condeelis JS

doi

10.2144/btn-2018-0112

subject

Has Abstract

pub_date

2019-03-01 00:00:00

pages

113-119

issue

3

eissn

0736-6205

issn

1940-9818

journal_volume

66

pub_type

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