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BACKGROUND
Assay fragmented DNA is essential to investigate the apoptosis. TUNEL assay is a method to detect fragmented DNA in apoptotic cells grown on the coverslip. Terminal deoxynucleotidyl transferase dUTP nick end labeling, is abbreviated as TUNEL.
Aim of this experiment is to perform TUNEL assay to find out cells with fragmented DNA fixed on the coverslip.
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REQUIREMENTS
Sample: Cells fixed on the coverslip.
Apparatus: 24-well plate and fluorescent microscope.
Chemicals: Paraformaldehyde (cell culture level),
1X HBSS,
TUNEL assay kit.
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PROCEDURE
Prior to harvest cells on the coverslip first coat it with gelatin or fibronectin. Get the adherent cells on the coverslip and carefully place in the 24-well plate. Wash with HBSS for 1-3 times per 5 min each, fixed with 4%- paraformaldehyde for minimum 2 hrs. Wash it with HBSS, take out the coverslip from the well, keep on the slides and add 10-20 µl of TUNEL mix and cover the section, keep at 37°C for one hour (before adding TUNEL mix, you can treat cells with 0.1% of triton X-1000. Wash with HBSS and add propidium iodide 50-100µl keep for 5 min at RT. Wash with HBSS for 3 times per 5 min. Mount with Hoechst dye stain dye keep in the shadow. You can take image in fluorescent microscope.1
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CONCLUSION
In this experiment, we learned about the TUNEL assay to detect cells having fragmented DNA.
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REFERENCES
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Sinha RA, Khare P, Rai A, Maurya SK, Pathak A, Mohan V, et al. Anti-apoptotic role of omega-3-fatty acids in developing brain: perinatal hypothyroid rat cerebellum as apoptotic model. Int. J. Devl Neuroscience. 2009;27(4):377–83.
