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

  •   FigureFigure 12.30.1 Expression efficiency increases with an increasing number of BacMam viral particles per cell (PPC). (A) Transduction efficiency was measured for HeLa cells transduced with a range of viral particles per cell. Three different BacMam targeted FPs (CellLight Tubulin‐GFP, CellLight Mitochondria‐RFP, CellLight Early Endosome‐RFP) were used. Increasing the number of BacMam viral particles increased the percentage of cells that were transduced. (B‐D) Increasing the expression can alter the subcellular localization of targeted fluorescent proteins. Typical localizations are shown for CellLight Tubulin‐GFP (BI), CellLight Mitochondria‐RFP (CI), and CellLight Early Endosome‐RFP (DI) at 40 particles per cell. Increasing the number of BacMam viral particles per cell to 100 causes aberrant localization of CellLight Tubulin‐GFP (BII), CellLight Mitochondria‐RFP (CII), and CellLight Early Endosome‐RFP (DII). Data in A were acquired using a BD Pathway 855 high‐content imaging platform. Images in B‐D were acquired using a Zeiss LSM 710 confocal microscope.
  •   FigureFigure 12.30.2 Transduction of primary and established cell lines with BacMam viruses. (A) Human cortical neuron cells (HCN) were transduced with CellLight ER‐GFP. (B) Normal rabbit kidney cells (RK‐13) transduced with CellLight Membrane‐ CFP. (C) Normal opossum kidney cortex proximal tubule cells (OK) transduced with CellLight Golgi‐GFP. (D) Human osteosarcoma cells (U2OS) transduced with CellLight MAP4‐GFP. (E) Normal fox lung fibroblasts (FoLu) transduced with CellLight Mito‐RFP. (F) Dual labeling with BacMam viruses: human osteosarcoma cells (U2OS) transduced with CellLight Tubulin‐GFP and CellLight Early Endosomes RFP. BacMam technology is ideal for transduction of primary cell lines that are difficult to transfect with other reagents.
  •   FigureFigure 12.30.3 pH effects on fluorescence intensity of pHrodo Red. (A) Relative increases of pHrodo dyes from nearly nonfluorescent state at neutral pH in cellular medium to increasingly higher fluorescence intensity through progressive stages of endocytosis. (B) Solution‐based emission profile for pHrodo Red at different pH levels, illustrating progressively higher peak emission at lower pH.
  •   FigureFigure 12.30.4 Labeling peroxisomes with minimal peroxisome targeting SKL‐GFP. HeLa cells were transduced with CellLight Peroxisomes‐GFP (A) Cells were counterstained with Hoechst 33342 to give total cell number (B). Images were acquired on a Delta Vision Core microscope.
  •   FigureFigure 12.30.5 Imaging autophagosomes with GFP‐tagged LC3B. HeLa cells were transduced with Premo Autophagy sensor GFP‐LC3B and left overnight. The following day, cells were washed three times with EBSS (supplemented with 2 mM MgCl2, 2 mM CaCl2, and 20 mM HEPES) and imaged on a Zeiss LSM 710 confocal microscope. (A) Each cell showed an increase in the number of vesicles present after 2 hr of starvation in EBSS. Image of HeLa cells at the beginning of starvation (BI) and after 2 hr (BII).
  •   FigureFigure 12.30.6 Effect of viral concentration on expression efficiency. Transduction was measured for HeLa cells transduced with a range of viral particles per cell (CellLight H2B‐RFP) in two different volumes (50 µl and 300 µl). Transduction efficiency is enhanced by reducing the volume of medium, thereby increasing the concentration of virus used for transduction.
  •   FigureFigure 12.30.7 Labeling endosomes using targeted fluorescent proteins. HeLa were transduced with CellLight Early Endosomes GFP (blue) and CellLight Late Endosomes RFP (red). The following day, cells were labeled with 50 nM MitoTracker Deep Red FM for 5 min at 37°C (green).
  •   FigureFigure 12.30.8 Measuring endocytosis with fluorogenic fluid‐phase markers. HeLa cells were incubated in 20 µg/ml pHrodo‐10k‐dextran for 30 min at 37°C. Cells were counterstained with Hoechst 33342 to give total cell number. Image acquired on a Delta Vision Core microscope.
  •   FigureFigure 12.30.9 Labeling peroxisomes with anti‐PMP70. HeLa cells fixed, permeabilized, and stained with rabbit monoclonal anti‐PMP‐70. Cells were counterstained with Hoechst 33342 to give total cell number. Image were acquired on a Delta Vision Core microscope.
  •   FigureFigure 12.30.10 Quantifying autophagy using anti‐LC3B. HeLa cells were treated with either vehicle (control) or 60 µM chloroquine and left overnight. The following day, cells were fixed and processed for ICC. Inhibition of autophagic flux by chloroquine causes an increase in autophagosomes (quantified by an increase in the intensity from vesicles around the nucleus). (B) Images showing control‐ (BI) or chloroquine‐ (BII) treated cells. Data were acquired on a BD Pathway 855 high‐content imager and a Thermo Fisher Arrayscan VTI.
  •   FigureFigure 12.30.11 Labeling of lysosomes using acidotropic dyes. A549 cells were stained with 400 nM LysoTracker Red DND‐99 (red) for 4 min at 37°C. Other cellular structures were labeled in the same cells using transduction of CellLight ER‐GFP (green, Life Technologies). Image acquired on a Delta Vision Core microscope.

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