TUNEL Assay

1. Prepare cells with test compounds

2. Incubate with TUNEL working solution for 30 min to 1 hour at 37 °C

3. Wash the cells
4. Fix cells with 4% formaldehyde (optional)

5. Read fluorescence intensity using fluorescence microscope with TRITC filter or flow cytometer with FL3 channel

Important  Thaw all the components at room temperature before starting the experiment.

Culture cells to an optimal density for apoptosis induction according to your specific protocol. We recommend about 30,000 to 50,000 cells/well for adherent cells grown in a 96-well microplate culture, or about 1 to 2 x 10⁶ cells/mL for non-adherent cells. At the same time, culture a non-induced negative control cell population at the same density as the induced population for every labeling condition. Note: We treated HeLa cells with 100 nM - 1 µM staurosporine for 4 hours to induce cell apoptosis. See Figure 1 for details.

For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html

Add 0.5 μL of 100X Tunnelyte™ Red (Component A) into 50 μL of Reaction Buffer (Component B) to make a total volume of 50.5 μL of TUNEL working solution. Protect from light. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density.

1. Remove cell media.
2. Add 50 µL of TUNEL working solution to each sample.
3. Incubate at 37°C for 30-60 minutes.
4. Remove TUNEL working solution, and wash the cells 1 - 2 times with 200 µL/well of PBS.
5. Add 100 uL Reaction buffer (Component B) to each sample.

6. Monitor the fluorescence intensity using a fluorescence microscope with TRITC filter or flow cytometer with FL3 channel.

7. Optional: Remove the reaction buffer from Step 5, and add 100 µL/well/96-well plate of 4% formaldehyde fixative buffer (not supplied) to each well. Note: For non-adherent cells, add desired amount (such as 2X10⁶ cells/mL) of 4% formaldehyde fixative buffer.

8. Incubate plates for 20 to 30 minutes at room temperature.
9. Remove fixative.
10. Wash the cells with PBS 2-3 times, and replace with 100 µL PBS/well/96-well plate.

11. Monitor the fluorescence intensity using a fluorescence microscope with TRITC filter or flow cytometer with FL3 channel.

12. Optional: Stain the nucleus with 1X Hoechst (Component C) at Ex/Em = 350/460 nm for image analysis