Journal Club—An In-Depth Look at Fluorescent Dyes for Organelle Labeling

• “Part I: BacMam labeling and reagents for vesicular structures” Dolman NJ, Kilgore JA, Davidson MW (2013) Curr Protoc Cytom 65:12.30.1–12.30.27.
• “Part II: Reagents for non-vesicular organelles” Kilgore JA, Dolman NJ, Davidson MW (2013) Curr Protoc Cytom 66:12.31.1–12.31.24.
• “Part III: Reagents for actin, tubulin, cellular membranes, and whole cell and cytoplasm” Kilgore JA, Dolman NJ, Davidson MW (2014) Curr Protoc Cytom 67:12.32.1–12.32.17.

Choosing a fluorescent reagent for imaging cellular structures and organelles in live or fixed cells can be daunting. Not only must the excitation and emission spectra of the reagent match your imaging instrument, but often you also need to choose from among a number of reagents—including organic dyes, drug conjugates, and fluorescent protein constructs—that target the same structure. Compatibility with the cells, with other probes in the same experiment, and with the treatments and protocols you are using can lead to the question: How do I choose one fluorescent probe over another?

Each fluorescent reagent comes with its own set of characteristics that may prove to be beneficial or detrimental for a particular assay or environmental condition. Once a reagent is chosen, validated protocols are not always available, optimizations are often required, and image examples can be hard to locate. Moreover, when a problem arises during the course of the experiments, identifying a solution can be difficult due to a lack of troubleshooting notes in product manuals or published references.

A three-part series of review articles that helps to clarify these issues has recently been published in Current Protocols in Cytometry. Authored by our R&D scientists Jason Kilgore and Nick Dolman, who have developed and tested many Molecular Probes organelle stains, as well as imaging guru Michael Davidson of Florida State University, this series pulls together a wealth of references and personal knowledge that covers the most common fluorescent reagents for nearly every cellular structure or organelle, focusing primarily on microscopic analysis.

Each article is broken into sections by target structure or organelle. The discussion includes a short history on the different reagents that have been used and their characteristics. A featured reagent is highlighted, along with a validated protocol for that reagent with an estimate on protocol time, a troubleshooting guide in table format, an image example, and relevant recipes for stock and final solutions.

In Part I the authors focus on the BacMam reagents, which have been used for transient transduction of fluorescent proteins targeted to specific cellular structures. They also discuss options for vesicular organelles such as endosomes (featuring pHrodo™ dextran conjugates), lysosomes (featuring the organic dye LysoTracker Red DND-99), peroxisomes (featuring the BacMam reagent CellLight Peroxisome-GFP, Figure 1), and autophagosomes (featuring another BacMam reagent, the Premo™ Autophagy Sensor GFP-LC3B).

Figure 1. (Figure 12.30.4 from “Part I”) Peroxisome staining in HeLa cells. HeLa cells were transduced with CellLight Peroxisome-GFP to transiently express a GFP fusion protein in peroxisomes. Cells were counterstained with Hoechst 33342 dye (blue, nuclei). Reprinted with permission from Current Protocols in Cytometry.

In Part II the authors discuss reagents for labeling the endoplasmic reticulum and nuclear membranes (featuring the ER-Tracker™ dyes), Golgi apparatus (featuring dye-labeled ceramides), mitochondria (featuring the organic dye MitoTracker Red CMXRos, Figure 2), and nucleoli (featuring the nucleic acid dye SYTO RNASelect™ Green). The best nucleus-selective dyes for live- and fixed-cell labeling are also highlighted.

Figure 2. (Figure 12.31.6 from “Part II”) Mitochondria and actin staining in a BPAE cell. A live BPAE (bovine pulmonary artery endothelial) cell was labeled with MitoTracker Red CMXRos (red, mitochondria) and then fixed, permeabilized, and labeled with Alexa Fluor 488 phalloidin (green, F-actin) and DAPI (blue, nuclei). Reprinted with permission from Current Protocols in Cytometry.

In Part III the authors describe reagents for actin (featuring Alexa Fluor phalloidin peptide conjugates, Figure 2), tubulin (featuring the drug conjugate Tubulin Tracker™ Green), cellular membranes (featuring plasma membrane labeling with fluorescent wheat germ agglutinin (WGA) conjugates, Figure 3), and whole-cell and cytoplasm reagents (featuring the organic dye CFSE, also called CFDA, SE).

Figure 3. (Figure 12.32.3 from “Part III”) Mitochondria and plasma membrane staining in HeLa cells. Live HeLa cells were transfected using pShooter™ vector pCMV/myc/mito/GFP and Lipofectamine 2000 Transfection Reagent, stained with the Alexa Fluor 594 conjugate of wheat germ agglutinin (WGA) (red, plasma membrane), and then imaged using confocal microscopy. Reprinted with permission from Current Protocols in Cytometry.

All three articles in this Current Protocols in Cytometry series—Part I, Part II, and Part III—are available through the Wiley Online Library.