One Step TUNEL Apoptosis Detection Kit (FITC)

1. Preparation of TUNEL working solution

Prepare the TUNEL working solution on ice and protected from light immediately before use:

2. Adherent cells

1. Wash cells twice with PBS for 5 min each.

2. Fix with 4% paraformaldehyde in PBS for 30 min at 4°C.

3. Wash twice with PBS for 5 min each.

4. Permeabilize with PBS containing 0.3% Triton X-100 for 5 min at room temperature.

5. Wash twice with PBS for 5 min each.

6. Add 50 μL TUNEL working solution per sample and incubate at 37°C for 60 min protected from light.

7. Wash three times with PBS. Counterstain/mount with DAPI-containing antifade mounting medium if desired and observe FITC green fluorescence by fluorescence microscopy.

3. Suspension cells

1. Collect approximately 2 × 10⁶ cells by centrifugation and wash twice with PBS for 5 min each.

2. Fix for 30 min at 4°C. Gentle shaking during fixation is recommended to reduce extensive cell clumping.

3. Wash twice with PBS for 5 min each.

4. Resuspend cells in permeabilization solution and incubate for 5 min at room temperature.

5. Wash twice with PBS for 5 min each.

6. Add 50 μL TUNEL working solution and incubate at 37°C for 60 min protected from light.

7. Wash with PBS and resuspend in an appropriate volume of PBS for FITC detection by flow cytometry, or prepare slides for fluorescence microscopy.

Note: For a negative control, omit TdT Enzyme. FITC-positive cells indicate apoptotic cells with DNA strand breaks.