Terminal dUTP Nick End-Labeling (TUNEL) of Barley Aleurone Protoplasts

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Tony Rook
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Terminal dUTP Nick End-Labeling (TUNEL) of Barley Aleurone Protoplasts

Please find the following protocol:

Terminal dUTP Nick End-Labeling (TUNEL) of Barley Aleurone Protoplasts

Overview:

Degradation of nuclear DNA can be examined by the TUNEL assay, which allows for in situ detection of nicked DNA and DNA breaks at the single cell level. This method is based on the enzymatic labeling within the cell of free 3'-OH groups at the ends of nicked DNA and the blunt ends of broken double-stranded DNA with fluorescein-labeled deoxyuridine. This protocol requires the use of the TUNEL kit from Roche Molecular Biochemicals titled In situ Cell Death Detection Kit, Fluorescein.

Procedure:

1. Let 1 g of purified protoplasts settle for 30 min.

2. Remove the supernatant.

3. Add 2 to 3 μl of protoplasts to 50 μl of Fixative on a 10-well Teflon-coated microscope slide.

4. Fix the protoplasts by incubating for 2 hr at room temperature.

5. Wash 3 times for 20 min each with 40 μl of Gamborg's B-5 Medium Plus Mannitol at room temperature.

6. Permeabilize the fixed cells by incubating in 20 μl of ice-cold Acetone for 2 min on ice.

7. Add 20 μl of PBS to the wells and transfer the slides to room temperature.

8. Wash the fixed protoplasts twice for 3 min each with 40 μl of PBS.

9. Perform the TUNEL reaction using the instructions included with the In situ Cell Death Detection Kit, Fluorescein.

As a positive control, treat some of the fixed and permeabilized protoplasts with 0.1 Units/ml of DNase I (Pharmacia Biotech) for 30 min at 37°C and subsequently wash three times with PBS for 5 min before adding the TUNEL reaction mix.

As a negative control, omit the Terminal Deoxynucelotidyl Transferase from some of the samples in the TUNEL reaction mix.

10. After the TUNEL reaction is complete, mount the protoplasts in Mountant to stain the DNA.

11. Analyze the specimens using an epifluorescent microscope and capture all images using the same exposure time.

12. Quantitative analysis of the intensity of TUNEL fluorescence can be performed using an epifluorescent microscope and acquiring images with a cooled charge-coupled device (CCD) camera.

Solutions:

Fixative
0.55% (v/v) Glutaraldehyde (CAUTION Biohazard!)
1000 to 1050 mmol/kg osmolarity Mannitol
3.7% (w/v) Paraformaldehyde (CAUTION Biohazard!)
Prepare in Gamborg's B-5 Medium, pH 3.5
20 mM CaCl2

Required Kit
In situ Cell Death Detection Kit, Fluorescein
(Roche Molecular Biochemicals)

Mountant
20 μg/ml 4,6-Diamidino-2-Phenylindole (DAPI)
50% (w/v) Glycerol
0.1% (w/v) N-Propylgallate

PBS
pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl

Gamborg's B-5 Medium Plus Mannitol
1,000 to 1,050 mmol/kg osmolarity Mannitol
Prepare in Gamborg's B-5 Medium, pH 3.5

Bioreagents and Chemicals:

Acetone
N-Propylgallate
DNase I
Mannitol
Glycerol
Sodium Phosphate, Dibasic
Potassium Chloride
Sodium Chloride
Potassium Phosphate, Monobasic
Calcium Chloride
DAPI
Gamborg's B5 Medium
Paraformaldehyde
Glutaraldehyde

Reference and Link:

1. Fath A, Bethke PC, and Jones RL. Barley aleurone cell death is not apoptotic - Characterization of nuclease activities and DNA degradation. Plant J. 1999; 20: 305-316.

http://www.bio.com/protocolstools/protocol.jhtml?id=p2064