GF488 One-Step TUNEL Cell Apoptosis Assay Kit (Green Fluorescence)
High-sensitivity detection of early-stage new markers; fully compatible with cells and tissue sections.
One-click copy of information
Instruction manual- Product Description
- Composition
- Precautions
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Product Storage:
Store at –20°C during transport and for storage. Fluorescent reagents must be protected from light. Valid for one year.
Product Overview:
The GF488 One‑Step TUNEL Apoptosis Assay Kit is used to detect DNA strand breaks in apoptotic cells. Its principle relies on the incorporation of a fluorescently labeled dUTP (488‑dUTP) at the 3′‑OH ends of fragmented DNA, catalyzed by terminal deoxynucleotidyl transferase (TdT). This labeling can then be visualized using a fluorescence microscope or a flow cytometer. The kit is suitable for assessing apoptosis in both tissue samples—paraffin‑embedded sections and frozen sections—and cell samples, including adherent and suspension‑cultured cells.
Instructions for Use
Cell fixation and permeabilization
1. For suspension cells or cell suspensions:
a. Collect 1–2 × 10^6 cells and wash once with PBS.
b. Fix at room temperature with 4% paraformaldehyde for 10–15 minutes.
c. Wash 1–2 times with PBS.
d. Resuspend the cells in 0.5 mL of permeabilization buffer (0.5% Triton X-100 in PBS) and incubate at room temperature for 5 minutes.
e. Wash twice with PBS.
2. For adherent cells (48- or 96-well plates) or cell smears:
a. Wash once with PBS.
b. Fix at room temperature with 4% paraformaldehyde for 10–15 minutes.
c. Wash 1–2 times with PBS.
d. Add 0.1–0.2 mL of permeabilization buffer (0.5% Triton X-100 in PBS) to each well and incubate at room temperature for 5 minutes.
e. Wash twice with PBS.
3. For paraffin sections:
a. De-wax in xylene for 10–15 minutes, then re-de-wax in fresh xylene for another 10–15 minutes. Follow with 5 minutes in absolute ethanol, 2 minutes in 90% ethanol, 2 minutes in 80% ethanol, and 2 minutes in 70% ethanol. Finally, rinse three times in PBS, 5 minutes each time.
b. Wipe away any liquid around the tissue, then use a histology pen to draw a circle around the tissue. Add an appropriate volume of 20 μg/mL proteinase K (without DNase) into the circle, and incubate at room temperature for 15–30 minutes.
c. Wash three times with PBS. Note: Proteinase K must be thoroughly washed away to avoid interfering with subsequent reactions.
4. For frozen sections:
a. Fix with 4% paraformaldehyde at room temperature for 30–40 minutes.
b. Wash twice with PBS, 5 minutes each time.
c. Add permeabilization buffer (0.5% Triton X-100 in PBS) and incubate at room temperature for 5 minutes.
d. Wash twice with PBS.
Positive Control Sample Processing (Optional)
1. Dilute the 10× DNase I Buffer to 1× by mixing 9 parts deionized water with 1 part 10× DNase I Buffer. Add 50 μL of the 1× DNase I Buffer to the tissue or cells and allow it to equilibrate at room temperature for 5 minutes.
2. Prepare the DNase I reaction solution according to the table below:
1 sample 10 × DNase I Buffer 5 μl DNase I 2.5 μL ddH2O 42.5 μL Total volume 50 μL 3. Remove the 1× DNase I Buffer, add the DNase I reaction solution to the tissue or cells, and incubate at 37°C for 30 minutes (for a 48-well plate, place on a shaker at 200 rpm; for a 96-well plate, no shaker is required).
4. Wash 2–3 times with PBS.
TUNEL assay
Prepare an appropriate volume of TUNEL assay solution according to the table below; ensure thorough mixing and prepare fresh just before use.
1 sample TdT Enzyme 1 μL 5 × Equilibration Buffer 10 μL 488-dUTP 5 μL ddH2O 34 μL Total volume 50 μL 1. For adherent cells, cell smears, or tissue sections:
a. Remove the PBS, then add 50 μL of TUNEL reaction solution to each sample and incubate in the dark at 37°C for 60 minutes (for a 48-well plate, place on a shaker set to 200 rpm). Note: Adjust the volume of TUNEL reaction solution as needed based on the sample area. For tissue sections or cell smears, cover with a plastic slide after adding the TUNEL reaction solution to prevent evaporation.
b. Wash twice with PBS.
c. Nuclear staining (optional): Prepare an adequate volume of Hoechst 33342 staining solution by diluting Hoechst 33342 in PBS at a 1:500 ratio, and perform staining at room temperature in the dark for 5–10 minutes.
d. Wash twice with PBS.
e. Mount the slides and observe under a fluorescence microscope. The excitation and emission wavelengths for 488-dUTP are 491 nm and 512 nm, respectively. For Hoechst 33342, the excitation and emission wavelengths are 346 nm and 460 nm, respectively.
2. For suspension cells or cell suspensions:
a. Remove the PBS, add 50 μL of TUNEL reaction solution to each sample, vortex to mix, and incubate in the dark at 37°C for 60 minutes, vortexing every 15 minutes if desired.
b. Wash twice with PBS.
c. Nuclear staining (optional): Prepare an adequate volume of Hoechst 33342 staining solution by diluting Hoechst 33342 in PBS at a 1:500 ratio, and perform staining at room temperature in the dark for 5–10 minutes.
d. Wash twice with PBS.
e. Resuspend the cells in 300–500 μL of PBS.
f. Detect by flow cytometry or observe under a fluorescence microscope.
Self-supplied reagents
10 mM PBS, pH 7.2–7.6
4% paraformaldehyde solution (4% PFA)
Permeabilization buffer (0.5% Triton X-100 in PBS)
Proteinase K solution (20 μg/mL) (for tissue section analysis)
Reagents used for tissue section dehydration (xylene, graded ethanol)
Flow cytometry tube
48/96-well cell culture plate
Immunohistochemistry pen
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Product Composition
Item number/specification Component Name
DNase I
10 × DNase I Buffer
Volume: 25 μL
50 μL
100-131-20/50 (20T/50T) TdT Enzyme
5 × Equilibration Buffer 488-dUTP
Hoechst33342
20/50 μL
200/500 μL
100/250 μL
20/50 μL
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Precautions
1. Please thaw the reagents on ice; before opening, perform a brief centrifugation, and promptly return them to –20°C after use.
2. The experiment may include a positive control group; one positive control should be included per batch. Positive control cells are treated with DNase I, and this kit contains enough reagent for 10 positive controls.
3. It is recommended to include a negative control group in each experiment; the negative control should not contain TdT enzyme.
4. Enzyme preparations are relatively viscous, and the sample volume is small; therefore, proper pipetting techniques must be observed.
5. This kit is intended for research use only and is not for clinical diagnostic purposes.
Keywords
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