TUNEL assay cell type - A127, U87MG, U251MG, T98G human glioblastoma cells

TUNEL assay is the cell death detection method where the biochemical marker of apoptosis is DNA fragmentation. The assay involves the microscopical detection of generated DNA fragments with free 3'-hydroxyl residues. in apoptotic cells using enzyme terminal deoxynucleotidyl transferase (TdT) which adds biotinylated nucleotides at the site of DNA breaks. Major challenges of this method involve proper access of the enzyme which could be hampered by poor permeabilization and/or excessive fixation with cross-linking fixative (common with archival tissue). This issue can be resolved by optimizing the incubation time with Proteinase K or CytoninTM.

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4 years ago

4 years ago by Ercole Udinesi Italy

Are 10 week old samples in formalin still usable?

I have left my tumor samples in formalin for an extended period of time (around 10 weeks). Do you think I will be able to use them for TUNEL assay and get results? Thank you for your help in advance

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Upstream tips
Precoating slides with poly-L-lysine may enhance cell adherence.

Do not allow the samples to dry out.

All other FragELTM kit components, with the exception of the Mounting Media, should be kept on ice during usage then promptly returned to -20C
Protocol tips
Dilute 5X TdT Equilibration Buffer 1:5 with water.

Place slides in a humidified chamber and incubate at 37C for 1 - 1.5 hours after ading TdT Labeling Reaction Mixture
Protocol tips
Fix cell samples with Fixation solution for 1 h.

Incubate in Permeabilisation solution (0.1% Triton X-100 in PBS) for 2 min on ice.

Label and incubate for 60 min at +37°C in a humidified atmosphere in the dark
Downstream tips
If low labelling is found, reduce fixation time or try 2% buffered paraformaldehyde
Upstream tips
Grow cells without any growth factors for 24 h.

Treat cells with Cur or SLCP (25 μM) for 24–72 h
Protocol tips
fix cells with 4% paraformaldehyde for 15 min,
Downstream tips
View slides as soon as possible. Analyze cells within 3 hours of
Upstream tips
- 2 × 10^4 cells/well were seeded.

- Cells were pre-treated with ionizing radiation (5 Gy).
Protocol tips
- Cells were fixed with 4 % PFA for 20 min.

- Click-iT® reaction is done for 30 min and counter stained for another 30 min at room temperature.
Protocol tips
- Cells were primary rabbit monoclonal anti-human Survivin and rabbit polyclonal anti-mcalpain.
Downstream tips
- When there was a little or poor staining, optimise permeabilization step
by adjusting incubation time with permeabilization agent.
Upstream tips
Seed 2 × 10^5 cells/well
Protocol tips
For labeling incubate at 37˚C for 1 hour in a humidity chamber
Downstream tips
No staining in treated sample, Increase incubation time with Proteinase K or Cytonin.
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