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

BioTechniques

Orrin Stone¹  Robert J Eddy¹  Ved P Sharma²  John S Condeelis²  Vera DesMarais² 

[1] Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461, USA;;1Department of Anatomy &

关键词: actin;    cell motility;    F-tractin;    lamellipodia;    Lifeact;    phalloidin;   

DOI  :  10.2144/btn-2018-0112

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown