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.

中文翻译：

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使用多种肌动蛋白探针，固定方法和成像方式优化前沿F-肌动蛋白标记。

我们系统地评估了高度运动的乳腺腺癌细胞系中几种广泛使用的可商购的肌动蛋白丝探针的性能和可靠性，以优化富含F-肌动蛋白的动态片状脂蛋白的可视化。我们在三个成像平台的五个固定条件下评估了四个Phalloidin荧光团，两个抗肌动蛋白抗体和三个活细胞肌动蛋白探针，作为优化方案设计的基础。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随时间推移的荧光信号维持方面排名最高。肌动蛋白单克隆抗体的使用显示出在多种固定通透性条件下的显着局限性。