The Colorimetric TUNEL Apoptosis Assay Kit provides a highly sensitive, rapid, and straightforward method for detecting apoptotic cells. Following biotin-labeling and subsequent DAB development, apoptotic cells can be visualized under a standard light microscope.
Apoptosis is a form of programmed cell death, typically categorized into early, mid, and late stages based on characteristic cellular changes. Late-stage apoptosis is marked by alterations in nuclear morphology, including chromatin condensation, nuclear membrane degradation, and DNA fragmentation.
The principle of the TUNEL (TdT-mediated dUTP Nick-End Labeling) method for detecting cell apoptosis is as follows: When cells undergo apoptosis, some DNA endonucleases are activated, which cut the genomic DNA between nucleosomes. In normal or proliferating cells, there is almost no DNA breakage, so no 3'-OH is formed and few can be labeled. The exposed 3'-OH can be labeled with biotin-labeled dUTP under the catalysis of terminal deoxynucleotidyl transferase (TdT). Streptavidin-HRP labeled chains can bind to it. Through the catalysis of HRP, it can be detected by DAB color development, thus enabling the detection of cell apoptosis.
Kit Contents
T1456509 | Components | Appearance | 20 T | 50 T | Storage | T1456509A | TdT Enzyme | Liquid | 20 μL | 50 μL | -20℃. | T1456509B | Biotin-11-dUTP | Liquid | 40 μL | 100 μL | -20℃. | T1456509C | Reaction Buffer(5×) | Liquid | 200 μL | 500 μL | -20℃. | T1456509D | Streptavidin-HRP | Liquid | 20 μL | 50 μL | -20℃. Store in the dark. | T1456509E | DAB Solution A | Liquid | 1 mL | 12.5 mL | -20℃. Store in the dark. | T1456509F | DAB Solution B | Liquid | 1 mL | 12.5 mL | -20℃. Store in the dark. | T1456509G | DNase I (10U/μL) | Liquid | 10 μL | 25 μL | -20℃. | T1456509H | DNase I dilution solution | Liquid | 1 mL | 12.5 mL | -20℃. |
Note: The 20T packaging of this kit can test 20 slides, sections or 96-well plates, 48-well plates, 24-well plates or 12-well plates of samples; it can test 10 6-well plates of samples; the 50T packaging of this kit can test 50 slides, sections or 96-well plates, 48-well plates, 24-well plates or 12-well plates of samples; it can test 25 6-well plates of samples. |
Usage method
Materials to Be Supplied by the User
1. 1X PBS (pH 7.4)
2. 0.1% Triton X-100 (Prepared in 1X PBS)
3. 4% Paraformaldehyde (Prepared in 1X PBS)
4. 3% H₂O₂
5. ddH₂O
6. Hematoxylin
Experimental Procedure
1. Sample Preparation
A. Suspension Cells or Cell Suspensions
a) Cell Collection: Centrifuge at 400 g for 5 minutes to collect the cells (no more than 2 million cells). Wash twice with PBS.
b) Fixation: Resuspend cells in an adequate volume of 4% Paraformaldehyde and fix at 4°C for 30 min. Centrifuge at 400 g for 5 min. Wash twice with PBS.
c) Permeabilization: Resuspend cells in an adequate volume of 0.1% Triton X-100 and incubate at room temperature (RT) for 15 min. Centrifuge at 400 g for 5 min. Wash twice with PBS.
d) Blocking: Add 100 μL of 3% H₂O₂ to cover the cell pellet. Incubate at RT in the dark for 20 min. Wash twice with PBS.
B. Adherent Cells or Cell Climbing Slides
a) Wash: Wash the cells once with PBS.
b) Fixation: Add an adequate volume of 4% Paraformaldehyde and fix at RT for 30 min. Wash twice with PBS.
c) Permeabilization: Add an adequate volume of 0.1% Triton X-100 and incubate at RT for 15 min. Wash twice with PBS.
d) Endogenous Peroxidase Blocking: Add 100 μL of 3% H₂O₂ to cover the cells. Incubate at RT in the dark for 20 min. Wash twice with PBS.
C. Paraffin-Embedded Tissue Sections
a) Deparaffinization and Hydration: Incubate in xylene for 5-10 min. Repeat with fresh xylene. Then incubate sequentially in: 100% ethanol (5 min), 90% ethanol (2 min), 70% ethanol (2 min), and finally distilled water (2 min).
b) Permeabilization: Dilute Protease K (2 mg/mL) to 20 μg/mL in PBS. Apply 100 μL to cover the tissue section and incubate at 37°C for 15-30 min.
c) Wash: Wash the section three times with PBS, 5 min each.
d) Blocking: Apply an adequate volume of 3% H₂O₂ and incubate at RT for 20 min.
e) Wash: Wash the section twice with PBS, 5 min each. Carefully remove excess liquid, ensuring the sample remains hydrated.
D. Frozen Tissue Sections
a) Fixation: Immerse sections in 4% Paraformaldehyde and fix at RT for 30 min. Rinse gently three times with PBS, 5 min each.
b) Permeabilization: Dilute Protease K (2 mg/mL) to 20 μg/mL in PBS. Apply 100 μL to cover the tissue section and incubate at 37°C for 10 min.
c) Wash: Rinse sections three times with PBS, 5 min each.
d) Blocking: Immerse sections in 3% H₂O₂ and incubate at RT for 20 min.
e) Wash: Rinse sections three times with PBS, 5 min each. Carefully dry around the section and use a hydrophobic pen to circle the tissue.
E. Positive Control (Optional)
a) Dilute the 10× DNase I Dilution Solution to 1× using ddH₂O.
b) Dilute DNase I (10 U/μL) to 20 U/mL using the 1X DNase I Dilution Solution.
c) Apply 100 μL of 1X DNase I Dilution Solution to the prepared sample and incubate at RT for 5 min for equilibration.
d) Remove the solution, apply 100 μL of diluted DNase I (20 U/mL), and incubate at RT for 10 - 30 min.
e) Remove DNase I solution and wash the sample twice with PBS.
2. TUNEL Reaction Mixture (Prepare Fresh)
Components of the TUNEL reaction solution | One sample | Five samples | Ten samples | TdT Enzyme | 1 μL | 5 μL | 10 μL | Biotin-11-dUTP | 2 μL | 10 μL | 20 μL | Reaction Buffer(5×) | 10 μL | 50 μL | 100 μL | ddH₂O | 37 μL | 185 μL | 370 μL | Total Volume | 50 μL | 250 μL | 500 μL |
Note: The biotin TUNEL reaction buffer and TdT Enzyme should be thoroughly dissolved and mixed before use. |
3. Biotin Labeling of Samples
A. Apply 50 μL of TUNEL Reaction Mixture to each sample, ensuring complete coverage. Incubate at 37°C for 60 min in a humidified chamber.
B. Remove the TUNEL Reaction Mixture. Wash three times with PBS (containing 0.1% Triton X-100 and 5 mg/mL BSA).
4. Color Development
A. Preparation of Streptavidin-HRP Working Solution:
Streptavidin-HRP working solution | One sample | Five samples | Ten samples | Streptavidin-HRP | 1 μL | 5 μL | 10 μL | PBS | 995 μL | 495 μL | 990 μL | Total Volume | 100 μL | 500 μL | 1000 μL |
Note: The prepared Streptavidin-HRP working solution must be used up completely at one time and should not be frozen. |
B. Preparation of DAB Coloring Solution
Prepare an appropriate amount of DAB color-developing solution by using 0.2 - 0.5 mL of the color-developing solution for each sample. Mix equal volumes of DAB Solution A and DAB Solution B, and after thorough mixing, it will be the DAB color-developing solution.
Note: The prepared DAB color-developing solution must be used up completely at one time and should not be frozen for storage.
C. Reaction: Add 100 μL of Streptavidin-HRP working solution onto the sample and incubate at room temperature for 30 minutes.
D. Washing: Discard the Streptavidin-HRP working solution and wash twice with PBS.
E. Color development: Add 0.2 - 0.5 mL of DAB color development solution and incubate at room temperature for 5 - 30 minutes or for an appropriate time depending on the color development.
Note: If the color development is strong, stop the color development within 5 minutes. If the color development is weak, extend the incubation time appropriately.
F. Termination: Discard the DAB color development solution and wash three times with PBS to terminate the color development reaction.
G. Cell nucleus staining (optional): Use hematoxylin staining solution or methyl green staining solution for cell nucleus staining.
H. Washing: Discard the cell nucleus staining solution and wash twice with PBS.
I. Observe directly, or dehydrate with 95% ethanol for 5 minutes, then dehydrate twice with 100% ethanol for about 3 minutes each time, then clear with xylene twice for 5 minutes each time, and then mount for observation.
Matters needing attention
1. Briefly centrifuge all vials prior to opening to bring the contents to the bottom.
2. For research use only. Not for use in diagnostic procedures.
3. Wear lab coats and disposable gloves for safety.
4. Sodium azide (NaN₃) is an inhibitor of HRP and must not be present in any solution.
5. Avoid repeated freeze-thaw cycles of the Biotin-11-dUTP and TdT Enzyme. Vortexing is not recommended.
6. Keep samples hydrated throughout the procedure to prevent drying artifacts.
