A Review of Reagents for Fluorescence Microscopy of Cellular Compartments and Structures, Part I: BacMam Labeling and Reagents for Vesicular Structures

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.

Keywords: autophagosomes; endosomes; fluorescent dyes; fluorescent proteins; lysosomes; peroxisomes.