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Identification of Neural Programmed Cell Death Through the Detection of DNA Fragmentation In Situ and by PCR

Yun C. Yung^1,2, Grace Kennedy¹, Jerold Chun¹

¹Helen L. Dorris Child and Adolescent Neuropsychiatric Disorder Institute, The Scripps Research Institute, La Jolla, California
²University of California, San Diego School of Medicine, San Diego, California

Publication Name:

Current Protocols in Neuroscience

Unit Number:

Unit 3.8

DOI:

10.1002/0471142301.ns0308s48

Online Posting Date:

July, 2009

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Abstract

Programmed cell death is a fundamental process for the development and somatic maintenance of organisms. This unit describes methods for visualizing both dying cells in situ and for detection of nucleosomal ladders. A description of various current detection strategies is provided, as well as support protocols for preparing positive and negative controls and for preparing genomic DNA. Curr. Protoc. Neurosci. 48:3.8.1-3.8.24. © 2009 by John Wiley & Sons, Inc.

Keywords: cell death; apoptosis; ISEL; TUNEL; nucleosomal ladder

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Introduction
Strategic Planning
Basic Protocol 1: Detection of Programmed Cell Death by In Situ End-Labeling Plus (ISEL+)
Support Protocol 1: Preparation of Thymocyte Cell Cultures for ISEL+
Basic Protocol 2: Detection of Nucleosomal Ladders Associated with Programmed Cell Death by Ligation-Mediated Polymerase Chain Reaction (LMPCR)
Support Protocol 2: Isolation of Genomic DNA from Tissues and Cultures
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Detection of Programmed Cell Death by In Situ End-Labeling Plus (ISEL+)

Materials

Pregnant mice or tissue culture cells grown on 12-mm glass coverslips coated with an appropriate adhesive (e.g., poly-l-lysine, appendix 2A, or Cell-Tak from Becton Dickinson)
DMEM or other dissection medium, 4°C
OCT compound (e.g., Tissue-Tek II from Sakura) or other water-soluble embedding medium, 4°C
Appropriate controls (see step 1 annotation)
Liquefied Histo-Freeze (Fisher Sci), CryoKwik (Damon), or liquid nitrogen
Dry ice, finely crushed (optional)
Fixative (see recipe)
2× SSPE (appendix 2A)
2× SSPE/0.6% Triton X-100 (see recipe)
0.1 M triethanolamine (TEA; see recipe)
Acetic anhydride (Sigma, cat. no. 91204)
DNase-free water
30%, 50%, 70%, 95%, and 100% ethanol, prepared with DNase-free water
ISEL+ TdT solution (see recipe)
Parafilm
MABS buffer (see recipe)
Blocking buffer (see recipe)
AP-conjugated sheep anti-DIG antibody (Roche, cat. no. 11093274910)
Alkaline phosphate buffer (see recipe)
Alkaline phosphatase color substrate buffer (see recipe)
Nuclear fast red solution (see recipe)
Permanent mounting medium (e.g., Crystal Mount from Biomeda and DPX from Fluka)
Unconjugated sheep anti-DIG antibody (Roche, cat. no. 11333089001)
Phosphate-buffered saline (appendix 2A)
Fluorescent tagged anti-sheep antibody (e.g., AlexaFluor 488 tagged; Invitrogen, cat. no. A-11015)
4¢,6-diamidino-2-phenylindole (DAPI; Sigma, cat. no. D9542)
Fluorescence mounting medium (e.g., Vectashield from Vector Labs)

Dissecting instruments including:
- Fine forceps
- Fine scissors
- Blunt forceps and spatula
Freezing molds (Fisher Scientific, optional)
Cryostat
Superfrost Plus glass slides (Fisher Scientific)
Slide-warmer (Barnstead Thermolyne), 50°C
Slide-processing holders and vessels
80°C vacuum oven with house vacuum/pump
24-well microtiter plates (if using coverslip-mounted cells as controls)
Airtight containers for slide storage with desiccant (Tupperware)
Humidified chamber for microscope slides
Coverslips
Microscope, preferably equipped for brightfield, DIC, and/or fluorescence

CAUTION: Paraformaldehyde, triethanolamine, acetic anhydride, and potassium cacodylate (in the TdT buffer) are toxic and must be used in accordance with safety standards.

Support Protocol 1: Preparation of Thymocyte Cell Cultures for ISEL+

Materials

4-week-old mice
DMEM/F-12 (appendix 2A), containing 0.0025% trypsin
DMEM/F-12/5% FBS (appendix 2A)
12-mm diameter round glass coverslips coated with poly-l-lysine (appendix 2A) or Cell-Tak (Collaborative Research)
Dexamethasone stock solution: 2 mM dexamethasone in 100% ethanol

Long-nose Pasteur pipet, flame polished
24-well microtiter dishes

Additional reagents and equipment for tissue culture (for cultured cells; see appendix 3B)

Basic Protocol 2: Detection of Nucleosomal Ladders Associated with Programmed Cell Death by Ligation-Mediated Polymerase Chain Reaction (LMPCR)

Materials

Isolated and quantified genomic DNA (see Support Protocol 2)
Oligonucleotides for ligation, unphosphorylated:
- 12-bp: 5¢-TGCGGTGAGAGG-3¢
- 24-bp: 5¢-AGCACTCTCGAGCCTCTCACCGCA-3¢
10× T4 DNA ligase buffer (prepared fresh; see recipe)
T4 DNA ligase (Roche; store up to 1 month at –20°C)
DNase-free water
PCR buffer (see recipe)
Oligonucleotides for single-copy-gene PCR controls: e.g., for mouse engrailed:
- 5¢-AGGACAAGCGGCCTCGCACA-3¢
- 5¢-CGGTGTCCGACTTGCCCTC-3¢
Taq DNA polymerase
Agarose gels, analytical grade (see appendix 1N), prepared in TBE buffer (appendix 2A)
Ethidium bromide staining solution (appendix 2A)

0.5-ml microcentrifuge tubes
Thermal cycler
Gel photographic setup

Additional reagents and equipment for agarose gel electrophoresis (appendix 1N)

CAUTION: Ethidium bromide is a mutagen and should be handled, stored, and disposed of with appropriate care.

Support Protocol 2: Isolation of Genomic DNA from Tissues and Cultures

Materials

Mice or tissue culture cells (see appendix 3B) grown on 12-mm glass coverslips coated with an appropriate adhesive (e.g., poly-l-lysine, appendix 2A, or Cell-Tak, Collaborative Research)
Sodium dodecyl sulfate (SDS)/proteinase K buffer (see recipe)
Molecular biology–grade 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol equilibrated with Tris×Cl, pH 8.0 (appendix 2A)
24:1 (v/v) chloroform/isoamyl alcohol
3 M sodium acetate, pH 5.0 (appendix 2A)
100% ethanol, –20°C
70% ethanol
TE buffer, pH 8.0 (appendix 2A)
RNase A solution, DNase-free (appendix 2A; optional)

Ceramic mortar and pestle, precooled with liquid nitrogen
15- and 50-ml polypropylene tubes with caps
50°C oven with rocker platform
Fluorometer or spectrophotometer

Additional reagents and equipment for DNA quantitation using a spectrophotometer (see appendix 1K)

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Figures

Figure 3.8.1

Diagram of workflow for Basic Protocol 1 and Basic Protocol 2 and time requirements.

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Figure 3.8.2

ISEL+ is more sensitive than TUNEL in identifying dying cells. Data are for thymi from 4-week-old mice treated with dexamethasone for 8 hr to induce massive PCD of cortical thymocytes, and then fixed and processed for ISEL+ or TUNEL. (A) A number of dying cells were identified by TUNEL in the cortex of the thymus (dark stain). (B) The ISEL+ procedure identified significantly more dying cells in the thymic cortex (dark stain). Scale bar = 20 µm.

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Figure 3.8.3

ISEL+ identifies dying cells earlier than TUNEL following the induction of PCD. Data are for thymic cultures prepared from 4-week-old mouse thymi, then treated with dexamethasone to induce PCD and fixed for either ISEL+ or TUNEL at various times after PCD treatment. Dying cells were identified by either the TUNEL (A, C, E) or ISEL+ (B, D, F) procedure. (A, B) 0 hr (control). Neither ISEL+ nor TUNEL identified many dying cells before dexamethasone treatment of cultures. (C, D) 0.5 hr. Although TUNEL (C) did not show an increase in dying cells, ISEL+ (D) detected an increase in cells undergoing PCD. (E, F) 4 hr. At this timepoint, TUNEL (E) detected an increase in dying cells produced by the dexamethasone treatment (at 1 hr and 2 hr there was still no increase in TUNEL-labeled cells; data not shown). Labeling looked similar to that shown by ISEL+ at 0.5 hr (D). ISEL+ labeling at 4 hr (F), however, showed a massive increase in dying cells, reflecting its much higher sensitivity. Scale bar = 20 µm.

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Figure 3.8.4

Labeling by ISEL+ is ~10 times more sensitive than TUNEL. Known quantities of a 1013-bp DNA fragment were labeled by either ISEL+ or TUNEL and electrophoresed on an agarose gel, then transferred to nitrocellulose and detected by ISEL+ or TUNEL. (A) TUNEL required 10 ng of labeled DNA per well for threshold detection. (B) ISEL+ detection of 10 ng labeled DNA is shown for comparison. This observation is consistent with thymic in situ data (see Fig. 3.8.2).

View Image
Figure 3.8.5

ISEL+ is specific in its identification of cells undergoing PCD. (A) ISEL+ identified relatively small numbers of dying cells in the cortex of the normal thymus, consistent with the normal rate of PCD in this tissue (dark stain). (B) After cortical thymocytes were induced to undergo PCD en masse by dexamethasone (Wyllie, 1981), ISEL+ labeled virtually all the thymocytes in the cortex. Scale bar = 50 µm. Abbreviations: ctx, thymic cortex; med, thymic medulla.

View Image
Figure 3.8.6

ISEL+ identifies dying cells in known models of neuronal PCD. Slides were viewed by ISEL+ (A, C, E) and DAPI counterstaining (B, D, F) for comparison. (A, B) P1 is the peak of PCD in the ganglion cell layer, and here >60% of cells were labeled by ISEL+. (C, D) ISEL+ identified dying motor neurons in the embryonic mouse (E14) spinal cord. (E, F). In the trigeminal ganglion at E16, dying neurons were easily identified by ISEL+ despite their low incidence at this age. Arrowheads indicate ganglion borders. Scale bar = 20 µm. Abbreviations: ret, retina; gcl, ganglion cell layer; sc, spinal cord; v, ventricle; tg, trigeminal ganglion.

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Figure 3.8.7

ISEL+ using AP and NPT/BCIP colorimetric detection is more sensitive than fluorescence detection. ISEL+ was run in parallel for identical lengths of time using both (A) AP and NPT/BCIP colorimetric detection and using (B) an AlexaFluor 488 (green) tagged secondary antibody on E12 mouse fetal liver to detect clusters of lymphocytes. The signal strength was stronger, the background was cleaner, and more dying cells were detected using colorimetric detection. Positive signal is denoted by dark stain and green fluorescence in the left and right panels, respectively. Scale bar = 100 µm.

View Image
Figure 3.8.8

Ligation-mediated PCR (LMPCR) identifies DNA nucleosomal ladders from tissues undergoing apoptosis. Mouse embryonic day E14 and adult brain tissue, as well as 4-week-old mouse thymus, were examined using LMPCR for the presence of nucleosomal ladder as an independent indication of apoptosis. Clear nucleosomal ladders can be seen for embryonic brain and adult thymus tissue, while adult brain showed significantly less intense laddering. A single-copy gene PCR control is also shown (for engrailed, en), confirming that equivalent amounts of genomic DNA were used for each sample. See Staley et al. (1997) for additional details.

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Literature Cited

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Key References
	Blaschke et al., 1996. See above.
First use of ISEL+ and the detection of programmed cell death in the embryonic cortex.
	Gavrieli et al., 1992. See above.
First technique to utilize labeling of DNA ends to detect programmed cell death.
	Raff et al., 1993. See above.
An informative discussion of nervous system programmed cell death.
	Staley et al., 1997. See above.
First use of ligation-mediated PCR to demonstrate apoptotic ladders in normal tissues.
	Wyllie, 1981. See above.
First demonstration of nucleosomal ladders associated with apoptosis.