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The ScienCellT Colorimetric TUNEL Apoptosis Assay is used for detection of apoptosis ��programmed cell death�� in individual cells based on the terminal deoxynucleotidyl transferase ��TdT��-mediated dUTP-biotin nick-end-labeling ��TUNEL�� technology. In brief, the 3��-OH end of the DNA strand breaks in apoptotic cells is labeled with biotinylated nucleotides using the enzyme TdT. Streptavidin conjugated horseradish peroxidase ��HRP�� is then bound to the biotinylated nucleotides, ��nd visualized using the peroxidase substrate, 3,3��-diaminobenzidine tetrachloride ��DAB��. The nuclei of apoptotic cells should be observed dark blue to bluish black under light microscope.

• PBS
• Paraformaldehyde
• Triton X-100
• Methanol
• H₂O₂
• Hematoxylin ��optional��
•  

The ScienCellT Colorimetric TUNEL Apoptosis Assay is applied to Human Astrocytes ��HAs�� treated with ��nd without DNase I, which serve as our positive ��nd negative controls, respectively. Dark nucleus can only be observed in cells treated with DNase I ��Figure 1��. The ScienCellT Colorimetric TUNEL Apoptosis Assay is also applied to Rat Cortical Neurons cultured with ScienCellT Neuronal Medium for 3 days, ��nd apoptotic cells can be identified with dark nucleus ��Figure 2��.

1. Cell culture
   1. Seed cells in 96-well culture plate in culture medium with or without test compounds. Culture the cells in a CO2 humidified incubator at 37°C for the desired period of time.
2. Pretreatment of cells
   1. Rinse cells three times with PBS, ��nd fix cells by incubating with freshly prepared 3.7% paraformaldehyde in PBS for 10 minutes at room temperature.
   2. Wash cells three times with PBS, for 2 minutes each time.
   3. Permeabilize cells by incubating with 0.2% Triton X-100 in PBS for 15 minutes at room temperature.
   4. Wash cells three times with PBS, for 2 minutes each time.
3. Setup of positive controls ��optional��
   1. Incubate positive control samples with DNase I ��100 µl per well�� for 10 minutes at room temperature to induce cleavage of genomic DNA.
4. TUNEL staining of cells
   1. Pre incubate cells with 100 µl/well of Equilibrium Buffer for 10 minutes at room temperature.
   2. Prepare TUNEL reaction mixture just before use based on the number of samples to be assessed. For each well of 96-well plate, mix 5 µl of TdT Solution with 45 µl of Biotin-dUTP Solution.
   3. Incubate cells in TUNEL reaction mixture ��50 µl per well�� for 60 minutes at 37ºC, protected from light.
   4. Wash cells three times with PBS, for 2 minutes each time.
   5. Block the endogeneous peroxidase activity by incubating cells with 100 µl per well of 3% H2O2 in methanol for 10 minutes at room temperature, protected from light.
   6. Wash cells three times with PBS, for 2 minutes each time.
   7. Prepare working streptavidin-HRP by diluting Stock Streptavidin-HRP 100× with PBS. Add 100 µl of working streptavidin-HRP to each well of 96-well plate, ��nd incubate for 30 minutes at 37ºC, protected from light.
   8. Wash cells three times with PBS, for 2 minutes each time.
   9. Prepare working DAB substrate by adding 5 ml of DI H2O to each vial of DAB Substrate. For best results use the solution immediately, otherwise aliquot ��nd store at -20°C. Add 100 µl of working DAB substrate to each well of 96-well plate, ��nd incubate for 1-5 minutes at room temperature in the dark, or until a blue background develop.
   10. Wash cells three times with PBS, for 2 minutes each time.
   11. Counter stain cells with hematoxylin if needed.
   12. Observe staining with a light microscope.

Storage

Store the basal medium at 4oC, the SGS, the FBS ��nd the P/S solution at -20°C. Protect from light.

Dry ice.

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