Differential Staining of DNA and RNA

Zbigniew Darzynkiewicz1, Gloria Juan1, Edward F. Srour2

1 New York Medical College, Valhalla, New York, 2 Indiana University School of Medicine, Indianapolis, Indiana
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.3
DOI:  10.1002/0471142956.cy0703s30
Online Posting Date:  November, 2004
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Abstract

Cell cycle analysis by means of differential staining of RNA and DNA permits determination of RNA content, which in turn allows one to discriminate G0 versus G1 cells and to detect cell differentiation. This unit presents two protocols for differential staining, one using the metachromatic dye acridine orange (AO) and the other a combination of pyronin Y (PY) and Hoechst 33342. Each method has its advantages and limitations, and the Hoechst‐PY method is not applicable to single‐laser instruments. A third protocol describes staining of viable cells to identify/sort hematopoietic stem cells, with an alternative that includes simultaneous immunostaining.

Keywords: flow cytometry; cell cycle analysis; nucleic acid staining; acridine orange; DNA staining; RNA staining; hematopoietic stem cells; pyronin Y; Hoechst 33342

     
 
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Table of Contents

  • Basic Protocol 1: Differential Staining of DNA and RNA of Unfixed Cells with Acridine Orange
  • Alternate Protocol 1: Differential Staining of Fixed Cells with Acridine Orange
  • Basic Protocol 2: Differential Staining of DNA and RNA with Hoechst 33342 and Pyronin Y
  • Support Protocol 1: Determination of Specificity of Cell Staining
  • Basic Protocol 3: Staining of Viable Cells with Hoechst 33342 and Pyronin Y to Identify/Sort Hematopoietic Stem Cells
  • Alternate Protocol 2: Simultaneous Cell Surface and Hoechst 33342 and Pyronin Y Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Differential Staining of DNA and RNA of Unfixed Cells with Acridine Orange

  Materials
  • Cells to be stained ( appendix 3B): ≤106 cells/ml suspended in tissue culture medium containing 10% (v/v) serum or 1% (w/v) BSA
  • Cell permeabilizing solution (see recipe), ice cold
  • Acridine orange (AO) staining solution (see recipe), ice cold
  • Flow cytometer equipped either with a 488‐ or 457‐nm argon‐ion laser (or both lasers) or with a mercury arc lamp
NOTE: In performing the following steps, carefully avoid pipetting, vortexing, or any mechanical agitation of cells, to prevent cell lysis.

Alternate Protocol 1: Differential Staining of Fixed Cells with Acridine Orange

  • Cells to be stained
  • PBS ( appendix 2A), ice cold
  • 70% ethanol, ice cold
  • Centrifuge, 4°C
  • Additional reagents and equipment for trypsinizing adherent cells (unit 5.2 or appendix 3B) or dissociating cells from tissues (unit 5.2)

Basic Protocol 2: Differential Staining of DNA and RNA with Hoechst 33342 and Pyronin Y

  Materials
  • Cells to be stained
  • PBS ( appendix 2A), ice cold
  • 70% ethanol, ice cold
  • Hanks' balanced salt solution (HBSS) containing Mg2+ and Ca2+ ( appendix 2A), ice cold
  • Pyronin Y (PY)–Hoechst 33342 staining solution (see recipe), ice cold
  • Centrifuge, 4°C
  • Flow cytometer equipped either with two lasers or with one laser and a mercury arc lamp
  • Additional reagents and equipment for trypsinizing adherent cells (unit 5.2 or appendix 3B) or dissociating cells from tissues (unit 5.2)

Support Protocol 1: Determination of Specificity of Cell Staining

  • Cells fixed in ethanol (see protocol 2, step , or see protocol 3, step )
  • RNase solution: 100 µg/ml RNase A in PBS containing Mg2+
  • DNase solution: 500 µg/ml DNase I in PBS containing Mg2+

Basic Protocol 3: Staining of Viable Cells with Hoechst 33342 and Pyronin Y to Identify/Sort Hematopoietic Stem Cells

  Materials
  • Viable cells to be stained
  • Hoechst staining buffer (see recipe)
  • Hoechst 33342 working solution (see recipe)
  • Pyronin Y working solution (see recipe)
  • 15‐ml snap‐cap tubes
  • Flow cytometer

Alternate Protocol 2: Simultaneous Cell Surface and Hoechst 33342 and Pyronin Y Staining

  • Surface immunostain (e.g., FITC‐labeled anti‐CD34, anti‐CD38, or anti‐CD133)
  • Isotype control
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Figures

  •   FigureFigure 7.3.1 Differential staining of RNA and DNA. Lymphocytes were analyzed (A) 0 hr, (B) 16 hr, (C) 24 hr, and (D) 48 hr after stimulation with phytohemagglutinin (PHA). The cells were stained with AO according to . Insets represent DNA content frequency histograms (green fluorescence) of the respective cell populations.
  •   FigureFigure 7.3.2 Differential staining of RNA and DNA with PY–Hoechst 33342 as described in .

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Literature Cited

Literature Cited
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   Crissman, H.A., Darzynkiewicz, Z., Tobey, R.A., and Steinkamp, J.A. 1985. Correlated measurements of DNA, RNA and protein in individual cells by flow cytometry. Science 228:1321‐1324.
   Darzynkiewicz, Z. 1988. Cellular RNA content, a feature correlated with cell kinetics and tumor prognosis. Leukemia 2:777‐787.
   Darzynkiewicz, Z. and Kapuscinski, J. 1990. Acridine orange: A versatile probe of nucleic acids and other cell constituents. In Flow Cytometry and Cell Sorting, 2nd ed. (M.R. Melamed, T. Lindmo, and M.L. Mendelsohn, eds.) pp. 291‐314. Wiley‐Liss, New York.
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