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

Nick J. Dolman1, Jason A. Kilgore1, Michael W. Davidson2

1 Molecular Probes Labeling and Detection, Life Technologies, Eugene, Oregon, 2 National High Magnetic Field Laboratory and Department of Biological Science, Florida State University, Tallahassee, Florida
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.30
DOI:  10.1002/0471142956.cy1230s65
Online Posting Date:  July, 2013
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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 allowed 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 for each structure, recommended protocol, troubleshooting guide, and example image. Curr. Protoc. Cytom. 65:12.30.1‐12.30.27. © 2013 by John Wiley & Sons, Inc.

Keywords: vesicles; autophagosomes; endosomes; peroxisomes; lysosomes; labeling; imaging; fluorescent dyes; fluorescent proteins; BacMam

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: BacMam Constructs
  • Alternate Protocol 1: Non‐Pseudo‐Typed BacMam Viruses/Hard to Transduce Cell Types
  • Basic Protocol 2: Labeling Endosomes: pHrodo‐10k‐Dextran
  • Basic Protocol 3: Labeling Peroxisomes: BacMam 2.0 CellLight Peroxisomes‐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: Performing Autophagosome Labeling with an Antibody
  • Basic Protocol 5: Labeling Lysosomes: LysoTracker Red DND‐99
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: BacMam Constructs

  Materials
  • Cell line/type of choice
  • Complete growth medium for selected cell line
  • High‐titer stocks (typically 1 × 108 pfu/ml) of BacMam virus (see recipe for BacMam reagents) containing construct to express the fluorescent protein of interest, stored at 4°C
  • Optional: 4% formaldehyde (made from methanol‐free, EM‐grade 16% formaldehyde) in DPBS (see recipe), HBSS (see recipe), or culture medium
  • Glass‐bottom Petri dishes, coverslips, or multi‐well plates appropriate for cell line
  • Method for counting cells (e.g., hemacytometer or Countess cell counter from Life Technologies)
  • Fluorescence microscope with filters appropriate for the fluorescent protein(s) being used

Alternate Protocol 1: Non‐Pseudo‐Typed BacMam Viruses/Hard to Transduce Cell Types

  • BacMam 1.0 or 2.0 virus (for extremely difficult‐to‐transduce cell types), at 1 × 108 pfu/ml (see recipe for BacMam construct)
  • Dulbecco's phosphate‐buffered saline (DPBS; see recipe)
  • BacMam Enhancer (optional; Life Technologies; see recipe for BacMam construct): 1000× concentration in DMSO

Basic Protocol 2: Labeling Endosomes: pHrodo‐10k‐Dextran

  Materials
  • Cell line/type of choice
  • Appropriate culture medium, phenol red–free
  • Hanks balanced salt solution (HBSS; see recipe) supplemented with 20 mM HEPES
  • 20 mg/ml pHrodo‐10k‐dextran (see recipe)
  • Glass‐bottom Petri dishes, coverslips, or multi‐well plates appropriate for cell line
  • Fluorescence microscope with filters for pHrodo‐10k‐dextran
  • Additional reagents and equipment for drug treatment or gene manipulation

Basic Protocol 3: Labeling Peroxisomes: BacMam 2.0 CellLight Peroxisomes‐GFP

  Materials
  • Cell line/type of choice
  • Complete growth medium for selected cell line
  • 1 × 108 pfu/ml CellLight Peroxisome‐GFP BacMam 2.0 (see recipe for CellLight BacMam 2.0 reagents)
  • Optional: 4% formaldehyde (made from methanol‐free, EM‐grade 16% formaldehyde) in DPBS (see recipe) or culture medium
  • Glass‐bottom Petri dishes, coverslips, or multi‐well plates appropriate for cell line
  • Fluorescence microscope with filter set for GFP

Alternate Protocol 2: Labeling Peroxisomes Using Antibodies

  Materials
  • Cell line/type of choice
  • Complete growth medium for selected cell line
  • 4% formaldehyde (made from methanol‐free, EM‐grade 16% formaldehyde) in
  • DPBS (see recipe) or culture medium (optional)
  • Dulbecco's phosphate‐buffered saline (DPBS; see recipe)
  • 0.2% to 0.5% Triton X‐100 in PBS (Life Technologies, cat. no. 10010)
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 10010)
  • 3% (w/v) bovine serum albumin (BSA) and 5% (v/v) heat‐inactivated normal goat serum (HINGS) in PBS (Life Technologies, cat. no. 10010)
  • Anti‐PMP70 primary antibody (Life Technologies, cat. no. 71‐8300)
  • Species‐specific (against species from which anti‐PMP70 antibody was derived) secondary antibody conjugated to a fluorescent dye
  • ProLong Gold antifade mounting medium (optional)
  • Glass‐bottom Petri dishes, coverslips, or multi‐well plates appropriate for cell line
  • Fluorescent microscope with filters specific for the dye conjugated to the secondary antibody

Basic Protocol 4: Labeling Autophagosomes: Transduction of Cells with Premo Autophagy Sensor GFP‐LC3B

  Materials
  • Cell line/type of choice
  • Complete growth medium for selected cell line
  • High‐titer stocks (typically 1 × 108 pfu/ml) of BacMam virus (see recipe) containing construct to express the fluorescent protein of interest, stored at 4°C
  • Premo Autophagy Sensor GFP‐LC3B BacMam 2.0 wild‐type (Life Technologies)
  • Premo Autophagy Sensor GFP‐LC3B mutant (G120A) (optional; Life Technologies)
  • Inducer of autophagy
  • Earle's buffered salt solution (EBSS; see recipe)
  • Glass‐bottom Petri dishes, coverslips, or multi‐well plates appropriate for cell line
  • Fluorescence microscope with filter set for GFP

Alternate Protocol 3: Performing Autophagosome Labeling with an Antibody

  Materials
  • Cell line/type of choice
  • Earle's buffered salt solution (EBSS; see recipe)
  • Inducer of autophagy, or inhibitor of autophagy (e.g., chloroquine diphosphate)
  • Optional: 4% formaldehyde (made from methanol‐free, EM‐grade 16% formaldehyde) in DPBS (see recipe) or culture medium
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 10010)
  • 0.2% to 0.5% Triton X‐100 in PBS (Life Technologies, cat. no. 10010)
  • 3% (w/v) bovine serum albumin (BSA) and 5% (v/v) heat‐inactivated normal goat serum (HINGS) in PBS (see recipe for DPBS)
  • Anti‐LC3B antibody (rabbit polyclonal LC3B from LC3 Antibody Kit for Autophagy; Life Technologies, cat. no. L10382)
  • Anti‐rabbit secondary antibody conjugated to a fluorescent dye
  • ProLong Gold antifade mounting medium (Life Technologies; optional)
  • Glass‐bottom Petri dishes, coverslips, or multi‐well plates appropriate for cell line
  • Fluorescent microscope with filters specific for the dye conjugated to the secondary antibody

Basic Protocol 5: Labeling Lysosomes: LysoTracker Red DND‐99

  Materials
  • 1 mM LysoTracker Red DND‐99 stock (see recipe)
  • Dulbecco's phosphate‐buffered saline (DPBS; see recipe), 37°C
  • Cell line/type of choice
  • Appropriate culture medium, phenol red–free
  • Optional: 4% formaldehyde (made from methanol‐free, EM‐grade 16% formaldehyde) in DPBS (see recipe) or culture medium
  • Fluorescence microscope with LysoTracker Red DND‐99 filters
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Figures

Videos

Literature Cited

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