Analysis of Intracellular Organelles by Flow Cytometry or Microscopy

Martin Poot1

1 University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.4
DOI:  10.1002/0471142956.cy0904s14
Online Posting Date:  May, 2001
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Abstract

Functional analysis of cellular organelles can be accomplished by staining cells with suitable organelle‐specific dyes and analyzing the fluorescence of the stained cells with a flow cytometer. With this methodology it is possible to resolve suspected heterogeneity in organelle function or content within a population of cells. Morphological information can be provided by quantitative microscopy (confocal microscope or video microscope with digital image‐analysis system). The thirteen protocols cover flow cytometry and microscopy for both live and fixed cells.

     
 
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Table of Contents

  • Mitochondria
  • Lysosomes
  • Golgi Apparatus
  • Endoplasmic Reticulum
  • Basic Protocol 1: Analysis of NAD(P)H Levels in Live Cell Mitochondria by Flow Cytometry
  • Basic Protocol 2: Analysis of Cardiolipin Levels in Live Cells
  • Basic Protocol 3: Analysis of Normalized Mitochondrial Membrane Potential
  • Basic Protocol 4: Analysis of Mitochondrial Oxidative Turnover in Live Cells
  • Basic Protocol 5: Leakage of Oxidant from Mitochondria
  • Basic Protocol 6: Staining of Live Cells for Measurement of Mitochondrial Mass or Function by Microscopy
  • Alternate Protocol 1: Mitochondrial Staining with Cell Fixation and Secondary Labeling
  • Basic Protocol 7: Staining of Live Cells for Measurement of Lysosomal Mass or Function by Flow Cytometry or Microscopy
  • Alternate Protocol 2: Staining of Lysosomal β‐Galactosidase Activity with FDG
  • Basic Protocol 8: Staining of Live Cells for Measurement of Golgi Apparatus Function by Microscopy
  • Alternate Protocol 3: Golgi Staining and Secondary Labeling Following Cell Fixation
  • Basic Protocol 9: Staining of Live Cells for Endoplasmic Reticulum Functional Analysis by Microscopy
  • Alternate Protocol 4: Endoplasmic Reticulum Staining Following Cell Fixation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of NAD(P)H Levels in Live Cell Mitochondria by Flow Cytometry

  Materials
  • Cells in suspension (see appendix 3B)
  • Cell culture medium supplemented with 10% FBS ( appendix 2A), 37°C
  • 15‐ml screw‐cap centrifuge tubes
  • 37°C water bath with cover
  • Flow cytometer with mercury arc lamp or argon laser (tuned to 360 nm) as excitation source
  • 12 × 75–mm polypropylene test tubes suitable for the flow cytometer
  • Computer for data collection and processing
  • Additional reagents and equipment for cell culture and harvesting ( appendix 3B)

Basic Protocol 2: Analysis of Cardiolipin Levels in Live Cells

  Materials
  • Cells in suspension (see appendix 3B)
  • Cell culture medium supplemented with 10% FBS ( appendix 2A), 37°C
  • 1 mM nonyl acridine orange dye stock solution (NAO; Table 9.4.1) in DMSO; store at −20°C in the dark
  • 15‐ml screw‐capped centrifuge tubes
  • 37°C water bath with cover
  • 12 × 75–mm polypropylene test tubes suitable for the flow cytometer
  • Flow cytometer with mercury arc lamp or argon laser (tuned to 488 nm) as excitation source
  • Band‐pass filter centered at ∼530 nm
  • 630‐nm long‐pass filter
  • Computer for data processing and collection
  • Additional reagents and equipment for cell culture and harvesting ( appendix 3B)
    Table 9.4.1   Materials   Key Features of Mitochondrial Dyes a   Key Features of Mitochondrial Dyes

    Dye Excitation maximum (nm) Emission maximum (nm) Excitation source b (nm) Collection wavelength c (nm) Sensitivity toward functional state Fixability with aldehydes Photostability d
    Rhodamine 123 506 530 488 (argon) 530 +
    MitoTracker Red CMXRos 594 608 488, 514 (argon) 543, 594 (HeNe) 610 +
    MitoTracker Green FM 480 516 488 (argon) 530 + +
    MitoFluor 480 516 488 (argon) 530 +
    Nonyl acridine orange 497 519 488 (argon) 530 +

     aFor further discussion, see Commentary.
     bOutput wavelengths of most commonly used flow cytometry lasers that are compatible with the excitation spectra of mitochondrial dyes.
     cFor flow cytometry, band‐pass filters should be centered around the indicated wavelength.
     d+, photographable with ordinary skill; −, difficult to photograph.

Basic Protocol 3: Analysis of Normalized Mitochondrial Membrane Potential

  Materials
  • Cells in suspension (see appendix 3B)
  • Cell culture medium supplemented with 10% FBS ( appendix 2A), 37°C
  • 200 µM MitoTracker Green FM (Table 9.4.1) dye stock solution in DMSO; store at −20°C in the dark
  • 200 µM CMXRosamine (Table 9.4.1) dye stock solution in DMSO; store at −20°C in the dark
  • 15‐ml screw‐cap centrifuge tubes
  • 37°C water bath with cover
  • 12 × 75–mm polypropylene test tubes suitable for the flow cytometer
  • Flow cytometer with mercury arc lamp or argon laser (tuned to 488 nm) as excitation source
  • Band‐pass filter centered at ∼530 nm
  • 630‐nm long‐pass filter
  • Computer for data collection and processing
  • Additional reagents and equipment for cell culture and harvesting ( appendix 3B)

Basic Protocol 4: Analysis of Mitochondrial Oxidative Turnover in Live Cells

  Materials
  • Cells in suspension (see appendix 3B)
  • Cell culture medium supplemented with 10% FBS ( appendix 2A), 37°C
  • 200 µM H 2‐CMXRosamine (Table 9.4.1) dye stock solution in DMSO; store at −20°C in the dark
  • 15‐ml screw‐cap centrifuge tubes
  • 37°C water bath with cover
  • 12 × 75–mm polypropylene test tubes suitable for the flow cytometer
  • Flow cytometer with mercury arc lamp or argon laser (tuned to 488 nm) as excitation source
  • 630‐nm long‐pass filter
  • Computer for data collection and processing
  • Additional reagents and equipment for cell culture and harvesting ( appendix 3B)

Basic Protocol 5: Leakage of Oxidant from Mitochondria

  Materials
  • Cells in suspension (see appendix 3B)
  • Cell culture medium supplemented with 10% FBS ( appendix 2A), 37°C
  • 10 mM 5‐(and‐6)‐carboxy‐2′,7′‐dichlorodihydrofluorescein diacetate stock solution in PBS; store at −20°C in the dark
  • 15‐ml screw‐cap centrifuge tubes
  • 37°C water bath with cover
  • 12 × 75–mm polypropylene test tubes suitable for the flow cytometer
  • Flow cytometer with mercury arc lamp or argon laser (tuned to 488 nm) as excitation source
  • Band‐pass filter centered at ∼530 nm
  • Computer for data collection and processing
  • Additional reagents and equipment for cell culture and harvesting ( appendix 3B)

Basic Protocol 6: Staining of Live Cells for Measurement of Mitochondrial Mass or Function by Microscopy

  Materials
  • Cells grown in monolayer ( appendix 3B) on coverslips
  • Cell culture medium supplemented with 10% FBS ( appendix 2A), 37°C
  • 9:1 (v/v) HBSS/FBS (freshly prepared; see appendix 2A for HBSS recipe)
  • Melted wax or nail polish
  • Mitochondrial dye stock solutions (see recipe): 0.5 mM stock solutions of rhodamine 123, dihydrorhodamine 123, MitoTracker Green FM, MitoFluor, nonyl acridine orange, or MitoTracker Red (CMXRos or CMXRos‐H 2)
  • 15 mM propidium iodide (aqueous; stable >1 year at 4°C) or 50 µM SYTOX Green working solution (prepare immediately before use from purchased stock solution)
  • 18 × 18–mm coverslips sterilized by dipping into absolute ethanol and subsequent flaming
  • 35‐mm cell culture dishes
  • Fluorescence microscope (unit 2.4)
  • Computer for data collection and processing
  • Additional reagents and equipment for cell culture and harvesting ( appendix 3B)
CAUTION: Due to the potential mutagenicity of propidium iodide and SYTOX Green (and other nucleic acid stains), it is preferable to purchase a concentrated stock rather than to prepare one.

Alternate Protocol 1: Mitochondrial Staining with Cell Fixation and Secondary Labeling

  • 3.7% formaldehyde (Fluka) in PBS (prepared fresh), 37°C.
  • PBS ( appendix 2A), 37°C
  • Anhydrous acetone (Sigma), −20°C
  • Additional reagents and equipment for immunocytochemistry (Watkins, )

Basic Protocol 7: Staining of Live Cells for Measurement of Lysosomal Mass or Function by Flow Cytometry or Microscopy

  Materials
  • Cells in suspension ( appendix 3B; for flow cytometry procedure) or grown in monolayer ( appendix 3B) on coverslips (for microscopy procedure)
  • Cell culture medium supplemented with 10% FBS ( appendix 2A), 37°C
  • 100 × LysoTracker Blue, Green, Yellow, or Red dye working solution (see recipe)
  • 15 mM propidium iodide (aqueous; stable >1 year at 4°C) or 50 µM SYTOX Green working solution (prepare immediately before use from purchased stock solution)
  • 9:1 (v/v) HBSS/FBS, 37°C (freshly prepared, for microscopy; see appendix 2A for HBSS recipe)
  • Melted wax or nail polish (for microscopy)
  • 15‐ml screw‐cap centrifuge tubes and 12 × 75–mm polypropylene tubes (for flow cytometry)
  • 18 × 18–mm coverslips sterilized by dipping into absolute ethanol and subsequent flaming (for microscopy)
  • 35‐mm cell culture dishes (for microscopy)
  • Flow cytometer with either a mercury arc lamp, an argon‐ion laser, or a HeNe laser as excitation source, or fluorescence microscope (unit 2.4)
  • Computer for data collection and processing
  • Additional reagents and equipment for cell culture and harvesting ( appendix 3B)
CAUTION: Due to the potential mutagenicity of propidium iodide and SYTOX Green (and other nucleic acid stains), it is preferable to purchase a concentrated stock rather than to prepare one.

Alternate Protocol 2: Staining of Lysosomal β‐Galactosidase Activity with FDG

  • 5 to 10 mM fluorescein di‐β‐D‐galactopyranoside (FDG) working solution (in culture medium; prepared fresh from purchased stock solution)

Basic Protocol 8: Staining of Live Cells for Measurement of Golgi Apparatus Function by Microscopy

  Materials
  • Cells grown in monolayer ( appendix 3B) on coverslips
  • Cell culture medium without pH indicator or serum, room temperature and ice‐cold
  • Ceramide‐BSA complex solution (see recipe)
  • Melted wax or nail polish
  • 18 × 18–mm coverslips sterilized by dipping into absolute ethanol and subsequent flaming
  • 35‐mm cell culture dishes
  • Fluorescence microscope (unit 2.4)
  • Additional reagents and equipment for adherent cell culture and harvesting ( appendix 3B)

Alternate Protocol 3: Golgi Staining and Secondary Labeling Following Cell Fixation

  • 3.7% formaldehyde (Fluka) in PBS (prepared fresh) or glutaraldehyde fixative solution (see recipe)
  • 0.5 mg/ml NaBH 4 (sodium borohydride) in unsupplemented cell culture medium (prepared fresh)
  • Defatted BSA solution (see recipe)
  • Additional reagents and equipment for immunocytochemistry (Watkins, )

Basic Protocol 9: Staining of Live Cells for Endoplasmic Reticulum Functional Analysis by Microscopy

  Materials
  • Cells grown in monolayer ( appendix 3B) on coverslips
  • Cell culture medium with and without 10% FBS ( appendix 3B), 37°C
  • 1 mM DiOC 6(3) stock solution (see recipe)
  • Melted wax or nail polish
  • 18 × 18–mm coverslips sterilized by dipping into absolute ethanol and subsequent flaming
  • 35‐mm cell culture dishes
  • Fluorescence microscope (unit 2.4)
  • Additional reagents and equipment for cell culture and harvesting ( appendix 3B)

Alternate Protocol 4: Endoplasmic Reticulum Staining Following Cell Fixation

  • Glutaraldehyde fixative solution (see recipe)
  • 0.5 mg/ml NaBH 4 (sodium borohydride) in unsupplemented cell culture medium (prepared fresh)
  • PBS ( appendix 2A)
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Figures

Videos

Literature Cited

Literature Cited
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