TUNEL assay cell type - HEK293 human embryonic kidney 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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5 years ago

5 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 2 matching solutions for this experiment

In Situ Cell Death Detection Kit, TMR red

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream 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	If low labelling is found, reduce fixation time or try 2% buffered paraformaldehyde

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

Manufacturer protocol Publication protocol 1 Relevant paper

ApopTag Fluorescein in Situ Apoptosis Detection Kit

Millipore

Upstream tips	Protocol tips	Downstream tips
	- Fix 5 x 10^7 cells/mL cells in 1% PFA in PBS, pH 7.4 - After applying Apply Equilibration Buffer and Working Strength TdT Enzyme , incubate at 37C for 1 hour.

Protocol tips
- Fix 5 x 10^7 cells/mL cells in 1% PFA in PBS, pH 7.4 - After applying Apply Equilibration Buffer and Working Strength TdT Enzyme , incubate at 37C for 1 hour.

Manufacturer protocol Publication protocol 1 Relevant paper

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