Department of Anatomy & Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461, USA.
Analytical Imaging Facility, Albert Einstein College of Medicine, Bronx, NY, USA.
Biotechniques. 2019 Mar;66(3):113-119. doi: 10.2144/btn-2018-0112.
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.
我们系统地评估了几种广泛使用的、商业上可获得的肌动蛋白丝探针在高度运动的乳腺腺癌细胞系中的性能和可靠性，以优化富含 F-肌动蛋白的动态片状伪足的可视化。我们在三个成像平台上的五种固定条件下评估了四种鬼笔环肽荧光染料、两种抗肌动蛋白抗体和三种活细胞肌动蛋白探针，为优化方案的设计提供了依据。在测试的荧光鬼笔环肽染料缀合物中，Alexa Fluor-488 鬼笔环肽在肌动蛋白细胞骨架的整体标记和荧光信号随时间的维持方面表现最佳。在各种固定-通透条件下，使用肌动蛋白单克隆抗体显示出明显的局限性。常用活细胞探针的评估提供了肌动蛋白丝偏向的证据，TagRFP-Lifeact 排除在片状伪足之外，但 mEGFP-Lifeact 或 F-肌动蛋白-EGFP 则不然。
