Abstract：
Fluorescent labeling of vesicular structures in cultured cells, particularly for live cells, can be challenging for a number of reasons. The first challenge is to identify a reagent that will be specific enough where some structures have a number of potential reagents and others very few options. The emergence of BacMam constructs has provided more easy-to-use choices. Presented here is a discussion of BacMam constructs as well as a review of commercially available reagents for labeling vesicular structures in cells, including endosomes, peroxisomes, lysosomes, and autophagosomes, complete with a featured reagent, recommended protocol, troubleshooting guide, and example image for each structure. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Delivering targeted fluorescent proteins using pre-made, high-titer BacMam constructs Alternate Protocol 1: Non-pseudo-typed BacMam viruses in standard cell types and pseudo-typed BacMam viruses in hard-to-transduce cell types Basic Protocol 2: Labeling endosomes: pHrodo™-10k-dextran Basic Protocol 3: Labeling peroxisomes: BacMam 2.0 CellLight™ Peroxisome-GFP Alternate Protocol 2: Labeling peroxisomes using antibodies Basic Protocol 4: Labeling autophagosomes: Transduction of cells with Premo™ Autophagy Sensor GFP-LC3B Alternate Protocol 3: Labeling autophagosomes using antibodies Basic Protocol 5: Labeling lysosomes: LysoTracker Red DND-99.
摘要：
培养细胞中囊泡结构的荧光标记，特别是对于活细胞，可能具有挑战性，原因有很多。第一个挑战是确定一种试剂，该试剂将具有足够的特异性，其中某些结构具有许多潜在的试剂，而其他结构具有很少的选择。BacMam结构的出现提供了更易于使用的选择。这里介绍了BacMam构建体的讨论以及用于标记细胞中囊泡结构的市售试剂的综述，包括内体，过氧化物酶体,溶酶体,和自噬体，配有特色试剂，推荐的协议，故障排除指南,和每个结构的示例图像。©2023威利期刊有限责任公司。基本方案1：使用预制的目标荧光蛋白，高滴度BacMam构建替代方案1：标准细胞类型中的非假型BacMam病毒和难转导细胞类型中的假型BacMam病毒基本方案2：标记内体：pHrodo™-10k-葡聚糖基本方案3：标记过氧化物酶体：BacMam2.0CellLight™过氧化物酶体-GFP使用Labsolyo细胞自噬方案2：标记DNA前抗体：标记3
