TUNEL assay cell type - SK-MEL-2 human melanoma

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.

3 Matching solutions
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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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Found 3 matching solutions for this experiment

APO-BrdU™ TUNEL Assay Kit, with Alexa Fluor™ 488 Anti-BrdU

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
- Cell were pretreated with 50 μM, 75 μM, and 100 μM of honokiol for 12, 24, 48, or 72 hours.	- Cells were fixed with 1% (w/v) paraformaldehyde in PBS and place on ice for 15 minutes	- If signal intensity is low increase the incubation time for the DNA-labeling reaction

Upstream tips
- Cell were pretreated with 50 μM, 75 μM, and 100 μM of honokiol for 12, 24, 48, or 72 hours.
Protocol tips
- Cells were fixed with 1% (w/v) paraformaldehyde in PBS and place on ice for 15 minutes
Downstream tips
- If signal intensity is low increase the incubation time for the DNA-labeling reaction

Manufacturer protocol Publication protocol 1 Relevant paper

In Situ Cell Death Detection Kit, Fluorescein

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
	- Cells were fixed with 4% paraformaldehyde for 20 min. - Permeabilized with PBS containing 0.1% Triton X-100 and 0.1% glycine for 2 min on ice	- When Nonspecific labelling is present, reduce concentration of TdT. - When high background is present reduce concentration of labelling mix by 50%

Protocol tips
- Cells were fixed with 4% paraformaldehyde for 20 min. - Permeabilized with PBS containing 0.1% Triton X-100 and 0.1% glycine for 2 min on ice
Downstream tips
- When Nonspecific labelling is present, reduce concentration of TdT. - When high background is present reduce concentration of labelling mix by 50%

Manufacturer protocol Publication protocol 1 Relevant paper

DeadEnd™ Fluorometric TUNEL System

Promega

Upstream tips	Protocol tips	Downstream tips
1065 cells/well	Transfect for 48h	- When there was a little or poor staining, optimise permeabilization step by adjusting incubation time with permeabilization agent. - When there is a loss of tissue from slides, decrease Proteinase K incubation time.

Upstream tips
1065 cells/well
Protocol tips
Transfect for 48h
Downstream tips
- When there was a little or poor staining, optimise permeabilization step by adjusting incubation time with permeabilization agent. - When there is a loss of tissue from slides, decrease Proteinase K incubation time.

Manufacturer protocol Publication protocol 1 Relevant paper

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