Click-iT™ TUNEL Alexa Fluor™ 488 Imaging Assay

Protocol tips

Upstream tips	Protocol tips	Downstream 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.	- Cells were counter stained for 1 min at room temperature, protected from light.

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.

Publication protocol

NCI-H460 cells were cultured in complete medium 48 h or until cells reached ~85 % confluency, rinsed once with PBS and serum-starved in RPMI-1640 with 1 % BSA for 2 h at 37 °C in a humidified atmosphere containing 5 % CO2. Cells were stimulated with recombinant human TRAIL/TNFSF10 (TRAIL) (R&D systems) or PBS as a control treatment as indicated in the figure legends. Apoptosis was assessed by cleaved caspase 8 protein levels via western blot analysis or by TUNEL assay. The TUNEL assay was performed using Click-It® TUNEL AlexaFluor488® Imaging Kit (Invitrogen) according to the manufacturer’s protocol. In brief, cells were fixed with 4 % paraformaldehyde in PBS at room temperature for 20 min and permeabilized with Triton X-100 (0.25 % in PBS) for an additional 15 min. The cells were then washed twice and incubated with 50 μL of terminal deoxynucleotidyl transferase reaction buffer (Component A) for 10 min at room temperature. The buffer was removed and the cells were incubated with TUNEL reaction mixture containing terminal deoxynucleotidyl transferase for 1 h in a humidified chamber at 37 °C for 1 h. Post treatment, cells were washed three times with 3 % BSA in PBS for 2 min each and then incubated with 50 μL of Click-iT reaction mixture (containing Alexa 488) for 30 min at room temperature, protected from light. The cells were then washed with 3%BSA in PBS and the nuclei were stained with Hoechst 33342 for 1 min at room temperature, protected from light. The coverslips were washed twice with PBS before mounting onto a slide with ProLong® Gold Antifade Mount (Life technologies). Cell monolayers were examined using an Olympus BMX-60 microscope equipped with a cooled CCD sensi-camera (Cooke, Auburn Hills, MI), and images were acquired using Slidebook software (Intelligent Imaging Innovation, Denver, CO). Fluorescence images were processed with ImageJ analysis software for the quantification of TUNEL-positive nuclei and total number of nuclei. The number of TUNEL-positive cells in three random (20×) fields was counted and divided by the total number of nuclei to determine the percentage of TUNEL-positive nuclei.

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Papers

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Click-iT™ TUNEL Alexa Fluor™ 488 Imaging Assay below.

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