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Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria

Boris Zhivotosky¹, Sten Orrenius¹

¹Karolinska Institute, Stockholm, Sweden, Sweden

Publication Name:

Current Protocols in Cell Biology

Unit Number:

Unit 18.3

DOI:

10.1002/0471143030.cb1803s12

Online Posting Date:

November, 2001

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Abstract

Apoptotic cells share a number of common features, such as phosphatidylserine (PS) exposure, cell shrinkage, chromatin cleavage, nuclear condensation, and formation of pyknotic bodies of condensed chromatin. Necrotic cells exhibit nuclear swelling, chromatin flocculation, loss of nuclear basophilia, breakdown of cytoplasmic structure and organelle function, and cytolysis by swelling. This unit describes some of the techniques most commonly used to detect cell death. A number of assays are used for characterizing and distinguishing apoptosis and necrosis. Morphological assays include trypan blue exclusion, differential staining, and Hoechst staining. Methods to detect chromatin cleavage include TUNEL assays for whole cells and paraffin sections, DNA fragmentation assays using whole cells, assays of total genomic DNA, analysis of DNA fragmentation by agarose gel electrophoresis, phenol extraction of DNA for analysis of fragmentation, a quantitative assay for DNA fragmentation, and detection of DNA fragmentation by pulsed-field gel electrophoresis. A protocol is also provided for Cytospin preparations from cell suspensions.

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Unit Introduction
Morphology Assays
Basic Protocol 1: Measurement of Cell Death by Trypan Blue Exclusion
Basic Protocol 2: Differential Staining of Cells
Basic Protocol 3: Hoechst Staining of Cells
Support Protocol 1: Cytospin Preparation of Cells for Analysis
Assays for Chromatin Cleavage
Basic Protocol 4: TUNEL Assay for DNA Fragmentation in Cells
Alternate Protocol 1: TUNEL Assay in Paraffin-Embedded Sections
Basic Protocol 5: Detection of DNA Fragmentation in Whole Cells
Alternate Protocol 2: Detection of DNA Fragmentation in Total Genomic DNA
Alternate Protocol 3: Simple Protocol for Detection of DNA Fragments
Alternate Protocol 4: Phenol Extraction of DNA Fragments for Agarose Gel Electrophoresis
Basic Protocol 6: Quantitative Assay of DNA Fragmentation
Basic Protocol 7: Detection of High-Molecular-Weight Chromatin Fragments by Pulsed-Field Agarose Gel Electrophoresis
Methods for Analysis of Caspase Proteolytic Activity
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Measurement of Cell Death by Trypan Blue Exclusion

Materials

Cell suspension to be assessed
2× PBS tablets, pH 7.2 to 7.4 (Sigma) or 2× PBS (appendix 2A)
0.2% (w/v) trypan blue (Sigma) in 2× PBS (store up to 1 to 2 months at 4°C)
Hemacytometer: improved Neubauer type (Karl Hecht; Baxter) or equivalent
Coverslips (e.g., Chance Propper)
Light microscope

Basic Protocol 2: Differential Staining of Cells

Materials

Cytospin preparations of cells (see Support Protocol 1)
100% methanol
Acid dye: 0.1% (w/v) eosin Y/0.1% (w/v) formaldehyde/0.4% (w/v) sodium phosphate dibasic/0.5% potassium phosphate monobasic
Basic dye: 0.4% (w/v) methylene blue-polychromed/0.4% (w/v) azure/0.4% (w/v) sodium phosphate dibasic/0.5% (w/v) monobasic potassium phosphate
DPX mountant (in solution; BDH)
Coverslips (e.g., Chance Propper)
Microscope slides (e.g., Menzel-Glaser)
Light microscope

Basic Protocol 3: Hoechst Staining of Cells

Materials

Cell suspension
1× and 2× PBS, pH 7.2 to 7.4 (from Sigma 2× PBS tablets, or see appendix 2A)
4% (w/v) paraformaldehyde (see recipe)
10 µg/ml Hoechst 33342 dye (Molecular Probes) in PBS (appendix 2A)
50/50 (v/v) PBS/glycerol
Microscope slides (e.g., Menzel-Glaser)
Coverslips (e.g., Chance Propper)
Fluorescent microscope
Additional reagents and equipment for Cytospin preparations (see Support Protocol 1)

Support Protocol 1: Cytospin Preparation of Cells for Analysis

Materials

Cell suspension
PBS (appendix 2A)
Cytospin centrifuge and cups (Shandon/Lipshaw)

Basic Protocol 4: TUNEL Assay for DNA Fragmentation in Cells

Materials

Cell suspension
100% methanol, –20°C
PBS, pH 7.2 to 7.4 (appendix 2A)
1% (v/v) formaldehyde
70% ethanol, ice cold
Terminal deoxyribonucleotidyltransferase (TdT; 25 U/ml) and 10× buffer (0.3 M Tris base/1.4 M sodium cacodylate, pH 7.2/1 mM DTT; e.g., Boehringer Mannheim)
25 mM CoCl₂
1 mM Bio-16-dUTP (e.g., Boehringer Mannheim)
Termination buffer: 300 mM NaCl/30 mM sodium citrate (e.g., Sigma or equivalent)
Staining buffer (e.g., Sigma; see recipe)
FACS fluid (e.g., Becton Dickinson; optional; for flow cytometry)
DPX mountant (BDH)
5 µg/ml propidium iodide in PBS (appendix 2A)
Flow cytometer (e.g., Becton Dickinson) and tubes; or fluorescent microscope
Coverslips (e.g., Chance Propper)
Microscope slide (e.g., Objektträger, Menzel-Glaser)

Alternate Protocol 1: TUNEL Assay in Paraffin-Embedded Sections

Additional Materials (also see Basic Protocol 4)

Paraffin-embedded tissue sections on slides
4% (w/v) paraformaldehyde (see recipe) or 4% formaldehyde, in PBS (appendix 2A)
96%, 90%, and 80% ethanol
Xylene
BSA (e.g., Sigma)
10 mM Tris×Cl, pH 8 (appendix 2A)
20 µg/ml proteinase K in 10 mM Tris×Cl (appendix 2A for Tris×Cl)
3% (v/v) methanol
2% (w/v) BSA in PBS (appendix 2A for PBS)
ExtrAvidin-peroxidase (Sigma) diluted 1:50 in PBS/1% BSA/0.5% Tween 20
3-Amino-9-ethylcarbazole (AEC)
Fluorescent microscope
Coverslips (e.g., Chance Propper)
Microscope slide (e.g., Objektträger, Menzel-Glaser)

Basic Protocol 5: Detection of DNA Fragmentation in Whole Cells

Materials

1× and 5× TBE buffer (see recipe), pH ~8.0 at room temperature (do not adjust pH)
SeaKem GTG agarose (FMC Bioproducts)
Cell suspension
50 mg/ml RNase A (see recipe)
4× DNA loading buffer: 4× TBE buffer (see recipe) containing 40% (w/v) sucrose and 0.25% (w/v) bromphenol blue (e.g., Sigma; store up to 2 to 3 weeks at 4°C)
Ultrapure agarose (Life Technologies)
10% (w/v) SDS (see recipe)
20 mg/ml proteinase K in water (store in aliquots up to 1 year at –20°C)
1× DNA loading buffer: 1× TBE buffer (see recipe) containing 10% (w/v) sucrose and 0.25% (w/v) bromphenol blue (e.g., Sigma)
DNA marker VI (pBR328 DNA cleaved with BglI and HinfI; Boehringer Mannheim), 1 µl in 20 µl of 1× DNA loading buffer
TE buffer, pH 8.0: 10 mM Tris×Cl/1 mM EDTA
10 mg/ml ethidium bromide (see recipe)
Boiling water bath or microwave oven
Gel electrophoresis apparatus: GNA-100 (Amersham Pharmacia Biotech), Buffer Puffer (Owl Scientific), or equivalent
Power supply (Power-Pac 300, Bio-Rad, or equivalent)
Shaker at 4°C
MacroVue UV Transilluminator (Hoefer Scientific Instruments) or equivalent
Photoman Polaroid gel documentation system (Hoefer Scientific Instruments) or equivalent

Alternate Protocol 2: Detection of DNA Fragmentation in Total Genomic DNA

Additional Materials (also see Basic Protocol 5)

Lysis buffer: 2 mM EDTA/100 mM Tris×Cl, pH 8.0/0.8% (w/v) SDS (store at room temperature)

Alternate Protocol 3: Simple Protocol for Detection of DNA Fragments

Additional Materials (also see Basic Protocol 5)

Lysis buffer, 4°C: 5 mM Tris×Cl, pH 8.0/20 mM EDTA/0.5% (v/v) Triton X-100
100% ethanol, –20°C
5 M NaCl (appendix 2A)
RNase T1/A stock (see recipe)
Vacuum lyophilizer (e.g., Hetovac, Heto-Holten)

Alternate Protocol 4: Phenol Extraction of DNA Fragments for Agarose Gel Electrophoresis

Additional Materials (also see Basic Protocol 5)

Lysis buffer (see Alternate Protocol 3), 4°C
100% ethanol, –20°C
5 M NaCl (appendix 2A)
RNase T1/A stock (see recipe)
Phenol, TE-saturated (see recipe)
24:1 (v/v) chloroform/isoamyl alcohol (store mixture at room temperature in a fume hood)
0.5% (w/v) SDS

Basic Protocol 6: Quantitative Assay of DNA Fragmentation

Materials

Cell suspension to be assessed
Lysis buffer (see Alternate Protocol 3), ice cold
10% (w/v) and 5% (w/v) trichloroacetic acid (TCA; keep at room temperature in dark flasks)
Diphenylamine reagent (see recipe)
10-ml conical glass tubes
Round-bottom glass tubes
Water bath, 100°C

Basic Protocol 7: Detection of High-Molecular-Weight Chromatin Fragments by Pulsed-Field Agarose Gel Electrophoresis

Materials

Cell suspension
Agarose buffer (for molds; see recipe)
SeaPlaque GTG low-melting-point agarose (FMC Bioproducts)
20 mg/ml proteinase K in water (store in aliquots up to 1 year at –20°C)
Proteinase buffer (for plugs; see recipe)
TE buffer, pH 8.0:10 mM Tris×Cl/1 mM EDTA
50 mM EDTA, pH 8.0 (appendix 2A)
SeaKem GTG agarose (FMC Bioproducts)
5× TBE buffer (see recipe)
DNA size pulse markers: chromosomes from Saccharomyces cerevisiae (225 to 2200 kbp) and a mixture of DNA HindIII fragments, DNA, and DNA concatemers (0.1 to 200 kbp; Sigma; supplied premade in syringe)
Gel leveling table
100-µl insert molds (Amersham Pharmacia Biotech), stored in 0.1 M HCl
12- or 24-well tissue culture plates
50°C incubator
100°C water bath or microwave oven
Pulsed-field gel electrophoresis system: vertical gel chamber with cooling elements (Protean II, Bio-Rad); horizontal gel chamber (HE 100B); power supply (PS 500 XT); and Switchback pulse controller (PC 500, Hoefer Scientific Instruments)
Thermostatic circulator Multi Temp III (Amersham Pharmacia Biotech)

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Figures

Figure 18.3.1

RAPI-DIFF staining of Cytospin preparation of HL-60 cells. Cells were treated with different concentrations of a cytotoxic drug to induce apoptosis or necrosis. (A) Untreated cells, (B) apoptotic cells, and (C) necrotic cells.

View Image
Figure 18.3.2

Gel electrophoresis of DNA from Jurkat cells treated with different concentrations of a cytotoxic drug. (A) Conventional gel: M, marker; lanes 1 to 3, DNA from apoptotic, untreated, and necrotic cells, respectively. The DNA ladder, when present, is a strong indicator of apoptosis. (B) Pulsed-field gel: MM, markers; lanes 1 to 3, DNA from untreated, apoptotic, and necrotic cells, respectively.

View Image

Literature Cited

Literature Cited
	Alnemri, E.S., Livingston, D.J., Nicholson, D.W., Salvesen, G., Thornberry, N.A., Wong, W.W., and Yuan, J. 1996. Human ICE/CED-3 protease nomenclature.Cell 87:171.
	Anand, R. and Southern, E.M. 1990. Pulsed field gel electrophoresis. In Gel Electrophoresis of Nucleic Acid: A Practical Approach, 2nd ed. (D. Rickwood and B.D. Hames, eds.) pp. 101-123. IRL Press, Oxford.
	Bowen, I.D. 1980. Techniques for demonstrating cell death. In Cell Death in Biology and Pathology (I.D. Bowen and R.A. Lockshin, eds.) pp. 379-444. Chapman & Hall, London–New York.
	Burton, K. 1956. A study of the condition and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62:315-323.
	Cryns, V. and Yuan, J. 1998. Proteases to die for. Genes Devel. 12:1551-1570.
	Darzynkiewicz, Z., Robinson, J.P., and Crisstman, H.A. (eds.) 1994. Methods Cell Biol., Vols. 41 and 42(Flow Cytometry: Part A and Part B, 2nd ed.). Academic Press, San Diego.
	Gavrieli, Y., Sherman, Y., and Ben-Sasson, S.A. 1992. Identification of programmed cell death in situ via specific labelling of nuclear DNA fragmentation. J. Cell Biol. 119:493-501.
	Kerr, J.F.R., Wyllie, A.H., and Currie, A.R. 1972. Apoptosis: A basic biological phenomenon with wide ranging implications in tissue kinetics. Br. J. Cancer 26:239-257.
	Koopman, G., Reutelingsperger, C.P.M., Kuijten, G.A.M., Keehnen, R.M.J., Pals, S.T., and van Oers, M.H.J. 1994. Annexin V for flow cytometric detection of phosphatidylserine expression on B-cells undergoing apoptosis. Blood 84:1415-1420.
	McGahon, A.J., Martin, S.J., Bissonnette, R.P., Mahboubi, A., Shi, Y., Mogil, R.J., Nishioka, W.K., and Green, D.R. 1995. The end of the (cell) line: Methods for the study of apoptosis in vitro. Methods Cell Biol. 46:153-185.
	Oberhammer, F., Fritsch, G., Scmied, M., Pavelka, M., Printz, D., Purchio, T., Lassman, H., and Schulte-Hermann, R., 1993a. Condensation of the chromatin at the membrane of an apoptotic nucleus is not associated with activation of an endonuclease. J. Cell Sci. 104:317-326.
	Oberhammer, F., Wilson, J.W., Dive, C., Morris, I.D., Hichman, J.A., Wakeling, A.E., Walker, R.A., and Sikorska, M. 1993b. Apoptotic death in epithelial cells: Cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J. 12:3679-3684.
	Pollak, A. and Ciancio, G. 1990. Cell cycle phase–specific analysis of cell viability using Hoechst 33342 and propidium iodide after ethanol preservation. Methods Cell Biol. 33:19-24.
	Robinson, J.P., Darzynkiewicz, Z., Dean, P.N., Dressler, L.G., Orfao, A., Rabinovitch, P.S., Stewart, C.S., Tanke, H.J., and Wheeless, L.L. 1999. Current Protocols in Cytometry. John Wiley & Sons, New York.
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	Thornberry, N.A., Rano, T., Peterson, E., Rasper, D., Timkey, T., Garcia-Calvo, M., Houtzager, V., Nordstrom, P., Roy, S., Vaillancourt, J., Chapman, K., and Nicholson, D. 1997. A combinatorial approach defines specificities of members of the caspase family and granzyme B: Functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272:17907-17911.
	Walker, P.R., Smith, C., Youdale, T., Leblamc, J., Whitfield, J.F., and Sikorska, M. 1991. Topoisomerase II–reactive chemotherapeutic drugs induce apoptosis in thymocytes. Cancer Res. 51:1078-1085.
	Wyllie, A.H. 1980. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284:555-556.
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