Analysis of Cellular DNA Content by Flow Cytometry

Zbigniew Darzynkiewicz1, Xuan Huang2, Hong Zhao1

1 Department of Pathology and Brander Cancer Research Institute, New York Medical College, Valhalla, New York, 2 Hematology/Oncology, Case Western Reserve University Cleveland, Ohio
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 5.7
DOI:  10.1002/cpim.36
Online Posting Date:  November, 2017
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Abstract

Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells based on DNA‐content differences for their subsequent culturing, is described. Also presented are methods for staining cell nuclei isolated from paraffin‐embedded tissues. Available algorithms are listed for deconvolution of DNA‐content‐frequency histograms to estimate percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content. © 2017 by John Wiley & Sons, Inc.

Keywords: cell cycle; DAPI; DNA ploidy; DNA index; Hoechst 33342; PI; propidium iodide; 4′6‐diamidino‐2‐phenylindole

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Analysis of Fixed Cells Stained with Propidium Iodide
  • Alternate Protocol 1: Analysis of Fixed Cells Stained with 4′,6‐Diamidino‐2‐ Phenylindole (DAPI)
  • Basic Protocol 2: DNA Content Analysis Utilizing Detergents and Proteases
  • Alternate Protocol 2: DNA Content Analysis Utilizing Detergents Only
  • Basic Protocol 3: Supravital Staining of DNA
  • Basic Protocol 4: DNA Content Analysis From Paraffin‐Embedded Samples
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Analysis of Fixed Cells Stained with Propidium Iodide

  Materials
  • 70% ethanol
  • Cells to be stained
  • Phosphate‐buffered saline (PBS; )
  • Propidium iodide (PI)/Triton X‐100 staining solution with RNase A (see recipe)
  • 12 × 75–mm centrifuge tubes, polypropylene or silanized preferred
  • Beckman TJ rotor or equivalent
  • Flow cytometer with 488‐nm argon ion laser, or xenon or mercury arc lamp with short‐pass BG12 filter (∼420‐nm cutoff)
  • >600‐nm long‐pass filter (optional)
  • DNA content frequency deconvolution software (e.g., Multicycle from Phoenix Flow Systems, ModFit from Verity Software)
  • Additional reagents and equipment for preparing cell suspensions for fixation (Darzynkiewicz & Juan, )

Alternate Protocol 1: Analysis of Fixed Cells Stained with 4′,6‐Diamidino‐2‐ Phenylindole (DAPI)

  Additional Materials (also see protocol 1)
  • DAPI/Triton X‐100 staining solution (see recipe)
  • Flow cytometer with UV light illumination source—e.g., laser tuned to UV light at 340 to 380 nm, or mercury arc lamp with UG1 short‐pass (390‐nm cutoff) excitation and band‐pass 420 ± 20‐nm emission filters

Basic Protocol 2: DNA Content Analysis Utilizing Detergents and Proteases

  Materials
  • Tissue sample (e.g., resected tumor)
  • Citrate/DMSO buffer (see recipe)
  • Internal DNA content standard (see recipe)
  • Cell lysis solution (see recipe)
  • Trypsin‐inactivating solution, ice cold (see recipe)
  • Propidium iodide (PI)/spermidine staining solution, ice cold (see recipe)
  • 0.5 × 25–mm (25‐G × 1‐in.) injection needles
  • 1‐ and 10‐ml disposable syringes
  • 38 × 12.5–mm polypropylene tubes with caps
  • 24‐ to 34‐μm nylon mesh (Small Parts)
  • Flow cytometer with 488‐nm argon ion laser fluorescence excitation source or mercury arc or xenon lamp with BG12 optical filter
  • >600 nm long‐pass filter
  • DNA content frequency deconvolution software (e.g., Multicycle from Phoenix Flow Systems, ModFit from Verity Software)
  • Additional reagents and equipment for counting cells in a hemacytometer ( ; Strober, )

Alternate Protocol 2: DNA Content Analysis Utilizing Detergents Only

  Additional Materials (also see Basic Protocols protocol 11 and protocol 32)
  • Cell suspension
  • DAPI/PIPES staining solution (see recipe)
  • Flow cytometer with UV light illumination source (e.g., laser tuned to 340 to 380 nm, mercury arc or xenon lamp with UG1 optical filter)
  • 470 ± 20‐nm band‐pass filter
  • >600‐nm long‐pass filter (for sulforhodamine 101 staining)

Basic Protocol 3: Supravital Staining of DNA

  Materials
  • Hoechst 33342 staining solution (see recipe)
  • Cells in tissue culture medium
  • Flow cytometer with UV light illumination source (e.g., mercury arc or xenon lamp with UG1 optical filter)
  • 470 ± 20‐nm band‐pass filter
NOTE: This protocol is predominantly used for sorting live cells; however, because sensitivity of cells to Hoechst 33342 varies depending on cell type, it is possible that cell viability and their cell cycle progression may be affected by the staining.

Basic Protocol 4: DNA Content Analysis From Paraffin‐Embedded Samples

  Materials
  • Paraffin‐embedded tissue blocks
  • Xylene or xylene substitute (e.g., Histo‐Clear, National Diagnostics)
  • 100%, 95%, 80%, 70% and 50% ethanol
  • Protease solution, fresh (see recipe)
  • 0.15 M NaCl
  • DAPI/phosphate staining solution, fresh (see recipe)
  • Microtome (e.g., Shandon Lipshaw)
  • 57‐μm nylon mesh in sheets (for filtering) and made into 2 × 2–cm bags by heat welding (Small Parts)
  • Rotary shaker
  • 1‐ to 2‐mm diameter marbles or glass balls
  • 5‐ and 15‐ml tubes
  • 37°C water bath with rotary shaker
  • Phase‐contrast or interference contrast (Nomarski optics) microscope Flow cytometer with UV light fluorescence excitation source
  • DNA content frequency deconvolution software (e.g., Phoenix Flow Systems Multicycle software)
  • Additional reagents and equipment for hematoxylin and eosin (H & E) staining (Hofman & Taylor, ), light microscopy (Keller & Watkins, ), and counting cells in a hemacytometer ( ; Strober, )
CAUTION: Xylene is toxic. Wear gloves and keep lids on jars. When possible, xylene should be substituted by less toxic reagents such as Histo‐Clear.NOTE: Store xylene and ethanol solution in 20‐ml aliquots in closed, xylene‐resistant, glass or plastic containers (e.g., Coplin jars or Erlenmeyer flasks).
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Figures

Videos

Literature Cited

Literature Cited
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