2.12. Terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) assay
This protocol is extracted from research article:
SIRT1 is involved in adrenocortical cancer growth and motility
J Cell Mol Med, Mar 2, 2021; DOI: 10.1111/jcmm.16317

Cells seeded onto glass coverslips were treated with sirtinol for 48 hours, washed with PBS and then fixed in 4% paraformaldehyde for 15 minutes at room temperature. The cells were subsequently washed with PBS and then soaked for 20 minutes in PBS containing 0.25% of Triton X‐100. After two washes with deionized water, slides were incubated with TdT (terminal deoxynucleotidyl transferase) enzyme and EdUTP (5‐ethynyl‐2'‐deoxyuridine 5'‐triphosphate) overnight at room temperature, using Click‐iT TUNEL Alexa Fluor Imaging Assay (Thermo Fisher Scientific). The next day cells were incubated with a solution containing Alexa Fluor azide for 30 minutes at room temperature and then stained with DAPI (4',6‐diamidino‐2‐phenylindole) solution in order to analyse the nuclear morphology. Cells were observed under a fluorescence microscope (Olympus BX41) with a 20X objective.

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