TUNEL assay cell type - A549, NCI-H460, H1299 human alveolar carcinoma

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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2 years ago

2 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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Found 6 matching solutions for this experiment

Protocol tips
Add Tunel mixture and incubate for 60 min at +37°C in a humidified atmosphere in the
dark
Downstream tips
If no specific labelling, reduce concentration of TdT by diluting it 1:2 up to 1:10 with TUNEL Dilution Buffer.

If high background, reduce concentration of labeling mix
to 50% by diluting with TUNEL Dilution Buffer
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
Protocol tips
- Cells were fixed with 4%PFA at RT for 20 min instead of 15min.

- Click-iT reaction mixture was done for 30 min at room temperature, protected from light.
Downstream tips
- Cells were counter stained for 1 min at room temperature, protected from light.
Upstream tips
- During the assay, cells were treated at different time intervals: 24, 48, and 72 hours.
Protocol tips
- Fixed with 1% (w/v) paraformaldehyde in PBS for 15 min
Downstream tips
- If signal intensity is low, increase incubation time for the DNA-labeling reaction
Downstream tips
- When there was a little or poor staining, optimise permeabilization step
by adjusting incubation time with permeabilization agent.
Upstream tips
- 1.5 × 106 cells were platted for culture.
Downstream tips
- No positive signal could be due to poor permeabilization with Triton X-100
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