TUNEL Apoptosis Assay (TUNEL)

Cat. No. CB017

(50 Tests)

Product Description

The 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, and 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.

Kit Components

Equilibrium Buffer, 5 ml

TdT Solution, 250 µl

Biotin-dUTP Solution, 2.5 ml

Stock Streptavidin-HRP (100×), 50 µl

DAB Substrate, Tablets

DNase I, 1 ml

Materials to be Supplied by the User

PBS

Paraformaldehyde

Triton X-100

Methanol

H₂O₂

Hematoxylin (optional)

Procedures (96-well plate)

Cell culture

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.

B.     Pretreatment of cells

1.      Rinse cells three times with PBS, and 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.

C.     Setup of positive controls (optional)

Incubate positive control samples with DNase I (100 µl per well) for 10 minutes at room temperature to induce cleavage of genomic DNA.

D.     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 0.3% 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, and 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 5ml of DI H₂O to each vial of DAB Substrate. For best results use the solution immediately, otherwise aliquot and store at -20 ºC. Add 100 µl of working DAB substrate to each well of 96-well plate, and 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.

Usage

TUNEL Apoptosis Assay is used to coat cell culture vessels in vitro. TUNEL Apoptosis Assay is for research use only. It is not approved for human or animal use, or application in clinical or in vitro diagnostic procedures.