产品介绍
显色法TUNEL细胞凋亡检测试剂盒(Colorimetric TUNEL Apoptosis Assay Kit)为您提供了一种高灵敏度又快速简便的细胞凋亡检测方法。对于待检测的细胞或组织样品，经过生物素标记和后续的DAB显色等步骤，即可在普通光学显微镜下观察到凋亡细胞。显色法TUNEL细胞凋亡检测试剂盒(Colorimetric TUNEL Apoptosis Assay Kit)为您提供了一种高灵敏度又快速简便的细胞凋亡检测方法。对于待检测的细胞或组织样品，经过生物素标记和后续的DAB显色等步骤，即可在普通光学显微镜下观察到凋亡细胞。
细胞凋亡是程序性细胞死亡的一种形式。根据程序性细胞死亡过程中的细胞变化，细胞凋亡通常分为早期、中期和晚期。细胞凋亡后期的特征是核形态、染色质凝集、核膜降解和DNA断裂的变化。
TUNEL (TdT-mediated dUTP Nick-End Labeling)法检测细胞凋亡的原理为：细胞在发生凋亡时，会激活一些DNA内切酶，剪断核小体间的基因组DNA，而正常的或正在增殖的细胞中几乎没有DNA的断裂，因而没有3'-OH形成，很少能够被标记。暴露的3'-OH可在末端脱氧核酸转移酶(TdT)的催化下加上生物素标记的dUTP，辣根过氧化物酶(HRP)标记的链霉亲和素(Streptavidin-HRP)可与之结合，在HRP的催化下通过DAB显色可检测到凋亡细胞，从而实现细胞凋亡检测。
另本试剂盒含即用型强效阳性对照--星形孢菌素 (Staurosporine)
- 经典可靠：星形孢菌素是国际公认的广谱蛋白激酶抑制剂，能高效诱导多种细胞系发生凋亡。
- 即开即用：试剂盒内已包含星形孢菌素阳性诱导剂，无需另行购买或配制阳性对照试剂。
- 文献广泛验证：0.1-5 μM星形孢菌素处理2-3小时即可诱导DNA断裂和细胞凋亡。
产品组分
C1524962 | 组件 | 物理外观 | 20 T | 50 T | 储存条件 | 单次测试使用量 |
C1524962A | TdT Enzyme | 液体 | 20 μL | 50 μL | -20℃. | 1 μL |
C1524962B | Biotin-11-dUTP | 液体 | 40 μL | 100 μL | -20℃. | 2 μL |
C1524962C | 反应缓冲液(5×) | 液体 | 200 μL | 500 μL | -20℃. | 10 μL |
C1524962D | Streptavidin-HRP | 液体 | 20 μL | 50 μL | -20℃. 避光保存. | 1 μL |
C1524962E | DAB Solution A | 液体 | 1 mL | 2.5 mL | -20℃. 避光保存. | 5 μL |
C1524962F | DAB Solution B | 液体 | 1 mL | 2.5 mL | -20℃. 避光保存. | 25 μL |
C1524962G | DNase I (10U/μL) | 液体 | 10 μL | 25 μL | -20℃. | 1 μL |
C1524962H | DNase I 稀释液 | 液体 | 1 mL | 2.5 mL | -20℃. | 50 μL |
C1524962I | 星形孢菌素 (5mM) | 液体 | 20 μL | 50 μL | -20℃ | 按需 |
使用方法
1. 实验材料(自备)
1) 1×PBS (pH 7.4)；
2) 0.1% Triton X-100(1×PBS配制)；
3) 4%多聚甲醛(1×PBS配制)；
4) 3% H₂O₂；
5) ddH₂O；
6) 苏木素。
2. 悬浮细胞或细胞悬液
1) 细胞收集：400 g离心5分钟收集细胞(不超过200万细胞)。PBS洗涤2次。
2) 固定：加入适量4%多聚甲醛重悬细胞，4℃固定30分钟。400 g离心5分钟收集细胞，PBS洗涤2次。
3) 通透：加入适量0.1% Triton X 100，室温通透15分钟。400 g离心5分钟收集细胞，PBS洗涤2次。
4) 封闭：每孔样本加入100 μL 3% H₂O₂覆盖细胞，室温避光孵育20分钟以灭活内源性过氧化氢酶。PBS洗涤2次。
3. 贴壁细胞或细胞爬片
1) 洗涤：用PBS洗涤细胞一次。
2) 固定：加入适量4%多聚甲醛，室温固定30分钟。PBS洗涤2次。
3) 通透：加入适量0.1% Triton X 100，室温通透15分钟。PBS 洗涤2次。
4) 封闭：每孔样本加入100 μL 3% H₂O₂覆盖细胞，室温避光孵育20分钟以灭活内源性过氧化氢酶。PBS洗涤2次。
4. 石蜡组织切片
1) 石蜡组织切片脱蜡与水化：二甲苯中脱蜡5-10分钟。换用新鲜的二甲苯，再脱蜡5-10分钟。无水乙醇5分钟。90%乙醇2分钟。70%乙醇2分钟，蒸馏水2分钟。
2) 通透：将蛋白酶K (P274341, 2 mg/mL)用PBS稀释至终浓度为20 μg/mL，每个样本滴加100 μL覆盖样本，37℃孵育15-30分钟。
注：孵育时间过长会增加组织切片在后续洗涤步骤中从载玻片上脱落的风险，过短则可能造成透性处理不充分，影响标记效率；不同组织的最佳作用温度和时间需自行摸索。
3) 洗涤：PBS洗涤3次，每次5分钟。
注：这一步必须把蛋白酶K洗涤干净，否则会严重干扰后续的标记反应。
4) 封闭：加入适量3% H₂O₂，室温孵育20分钟以灭活内源性过氧化氢酶。
5) 洗涤：PBS洗涤2次，每次5分钟。用滤纸吸去多余液体，保持样品湿润。
5. 冰冻组织切片
1) 固定：将切片样本浸入4%多聚甲醛中，室温固定30分钟。PBS缓慢漂洗3次，每次5分钟。
2) 通透：将蛋白酶K (2 mg/mL)用PBS稀释至终浓度为20 μg/mL，每个样本滴加100 μL覆盖样本，37℃孵育10分钟。
注：孵育时间过长会增加组织切片在后续洗涤步骤中从载玻片上脱落的风险，过短则可能造成透性处理不充分，影响标记效率；不同组织的最佳作用温度和时间需自行摸索。
3) PBS漂洗切片3次，每次5分钟。
4) 封闭：将切片样本浸入3% H₂O₂溶液，室温孵育20分钟，以灭活切片内源的过氧化氢酶。
5) PBS漂洗切片3次，每次5分钟，用滤纸吸干切片样本周围液体，用免疫组化笔圈好样本轮廓，以便下游标记。将处理好的切片放在湿盒中保持湿润。
注：若在后续实验操作中发现免疫组化笔画的轮廓圈被破坏，需及时补画。
6. 阳性对照(选做)
1) 将DNase I (10 U/μL)用DNase I 稀释液至终浓度为100 U/mL。
2) 加入50 μL DNase I 到已处理的样品上，室温孵育10分钟。
3) 弃掉DNase I，PBS洗涤2次。
7. TUNEL反应液(现配现用)
TUNEL反应液组分 | 1个样品 | 5个样品 | 10个样品 |
TdT Enzyme | 1 μL | 5 μL | 10 μL |
Biotin-11-dUTP | 2 μL | 10 μL | 20 μL |
反应缓冲液(5×) | 10 μL | 50 μL | 100 μL |
ddH₂O | 37 μL | 185 μL | 370 μL |
总体积 | 50 μL | 250 μL | 500 μL |
注：生物素TUNEL反应缓冲液和TdT Enzyme需完全溶解混匀后使用。
8. 样品生物素标记
1) 每个样品加入50 μL TUNEL反应液，使反应液覆盖样品。37℃孵育1小时。
注：50 μL生物素标记液适合涂片、切片或96孔板、48孔板、24孔板或12孔板的一个孔，如果是6孔板中的一个孔生物素标记液宜使用100 μL。孵育时注意防护防止工作液挥发。
2) 弃掉TUNEL反应液，用PBS (含0.1% TritonX-100 和5 mg/mL BSA)洗涤3次。
9. Streptavidin-HRP工作液和DAB显色液的配制
1) Streptavidin-HRP工作液的配制
Streptavidin-HRP工作液 | 1个样品 | 5个样品 | 10个样品 |
Streptavidin-HRP | 1 μL | 5 μL | 10 μL |
PBS | 99 μL | 495 μL | 990 μL |
总体积 | 100 μL | 500 μL | 1000 μL |
注：配制好的Streptavidin-HRP工作液必须一次使用完毕，不宜冻存。
2) DAB显色液的配制：按照每个样品使用0.2-0.5 mL显色液的比例配制适量DAB显色液。1mL PBS分别加入10 μL DAB Solution A和50 μL DAB Solution B，充分混匀后即为DAB显色液。
注：配制好的DAB显色液必须一次使用完毕，不宜冻存。
10. 显色反应
1) 反应：在样品上加100 μL Streptavidin-HRP工作液，室温孵育30分钟。
2) 洗涤：弃掉Streptavidin-HRP工作液，用PBS洗涤2次。
3) 显色：滴加0.2-0.5 mL DAB显色液，室温孵育5-30分钟或根据显色情况孵育适当时间。
注：如果显色很强可以短于5分钟即停止显色，如果显色很弱，可以适当延长显色时间。
4) 终止：弃掉DAB显色液，PBS洗涤3次以终止显色反应。
5) 细胞核染色(可选)：用苏木素染色液(G774770)或甲基绿染色液进行细胞核染色。
6) 洗涤：弃掉细胞核染色液，PBS洗涤2次。
7) 直接进行观察，或用95%乙醇脱水5分钟，再用100%乙醇脱水2次，每次约3分钟，再用二甲苯透明2次，每次5分钟，随后封片观察。
注意事项
1. 使用前请将产品瞬时离心至管底，再进行后续实验。
2. 本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品。
3. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
4. 叠氮化钠 (NaN₃) 会抑制 HRP，因此反应体系中不能含有叠氮化钠。
5. 荧光标记液和TdT Enzyme避免反复冻融，建议不要涡旋。
6. 实验过程中请保持样本的湿润，防止干片造成的实验失败。
图1 TUNEL染色用于检测凋亡细胞。对照组未见明显阳性染色；经 DNase I 处理的阳性对照组中可见大量呈棕色、具有断裂 DNA 的TUNEL阳性细胞核，验证了该TUNEL染色试剂盒的性能。此外，星形孢菌素 (Staurosporine) 处理组同样显示出明显的棕色TUNEL阳性细胞核，进一步证明该试剂盒可有效检测化学诱导剂引发的细胞凋亡。以上结果表明，TUNEL显色法在不同实验模型中均具有高灵敏度和可靠性。
Product Introduction
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.
Additionally, this kit includes a ready-to-use, highly potent positive control, Staurosporine
- Classic and reliable: Staurosporine is an internationally recognized broad-spectrum protein kinase inhibitor that efficiently induces apoptosis in a wide range of cell lines.
- Ready-to-use: The kit includes staurosporine as a positive inducer, eliminating the need to purchase or prepare additional positive control reagents separately.
- Extensively validated in the literature: Treatment with 1 µM staurosporine for 2-3 hours induces DNA fragmentation and apoptosis; in HeLa cells, treatment with 100 nM or 1 µM for 4 hours yields bright TUNEL‑positive signals.
Components
C1524962 | Components | Appearance | 20 T | 50 T | Storage | Quantity Per Test |
C1524962A | TdT Enzyme | Liquid | 20 μL | 50 μL | -20℃. | 1 μL |
C1524962B | Biotin-11-dUTP | Liquid | 40 μL | 100 μL | -20℃. | 2 μL |
C1524962C | Reaction Buffer(5×) | Liquid | 200 μL | 500 μL | -20℃. | 10 μL |
C1524962D | Streptavidin-HRP | Liquid | 20 μL | 50 μL | -20℃. Store in the dark. | 1 μL |
C1524962E | DAB Solution A | Liquid | 1 mL | 2.5 mL | -20℃. Store in the dark. | 5 μL |
C1524962F | DAB Solution B | Liquid | 1 mL | 2.5 mL | -20℃. Store in the dark. | 25 μL |
C1524962G | DNase I (10U/μL) | Solid | 10 μL | 25 μL | -20℃. | 1 μL |
C1524962H | DNase I dilution solution | Liquid | 1 mL | 2.5 mL | -20℃. | 50 μL |
C1524962I | Staurosporine (5mM) | Liquid | 20 μL | 50 μL | -20℃ | As needed |
Protocol
1. Materials to Be Supplied by the User
1) 1×PBS (pH 7.4).
2) 0.1% Triton X-100 (Prepared in 1×PBS).
3) 4% Paraformaldehyde (Prepared in 1×PBS).
4) 3% H₂O₂.
5) ddH₂O.
6) Hematoxylin.
2. Suspension Cells or Cell Suspensions
1) Cell Collection: Centrifuge at 400 g for 5 minutes to collect the cells (no more than 2 million cells). Wash twice with PBS.
2) 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.
3) 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.
4) 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.
3. Adherent Cells or Cell Climbing Slides
1) Wash: Wash the cells once with PBS.
2) Fixation: Add an adequate volume of 4% Paraformaldehyde and fix at RT for 30 min. Wash twice with PBS.
3) Permeabilization: Add an adequate volume of 0.1% Triton X-100 and incubate at RT for 15 min. Wash twice with PBS.
4) 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.
4. Paraffin-Embedded Tissue Sections
1) 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).
2) Permeabilization: Dilute Protease K (P274341, 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.
3) Wash: Wash the section three times with PBS, 5 min each.
4) Blocking: Apply an adequate volume of 3% H₂O₂ and incubate at RT for 20 min.
5) Wash: Wash the section twice with PBS, 5 min each. Carefully remove excess liquid, ensuring the sample remains hydrated.
5. Frozen Tissue Sections
1) Fixation: Immerse sections in 4% Paraformaldehyde and fix at RT for 30 min. Rinse gently three times with PBS, 5 min each.
2) 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.
3) Wash: Rinse sections three times with PBS, 5 min each.
4) Blocking: Immerse sections in 3% H₂O₂ and incubate at RT for 20 min.
5) Wash: Rinse sections three times with PBS, 5 min each. Carefully dry around the section and use a hydrophobic pen to circle the tissue.
6. Positive Control (Optional)
1) Dilute DNase I (10 U/μL) with DNase I dilution buffer to a final concentration of 100 U/mL.
2) Add 50 μL DNase I solution onto the pretreated sample, and incubate at room temperature for 10 minutes.
3) Remove DNase I solution and wash the sample twice with PBS.
7. 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.
8. Biotin Labeling of Samples
1) Apply 50 μL of TUNEL Reaction Mixture to each sample, ensuring complete coverage. Incubate at 37°C for 60 min in a humidified chamber.
2) Remove the TUNEL Reaction Mixture. Wash three times with PBS (containing 0.1% Triton X-100 and 5 mg/mL BSA).
9. Preparation of Streptavidin-HRP Working Solution and DAB Chromogenic Substrate
1) 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 | 99 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.
2) 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. Add 10 μL of DAB Solution A and 50 μL of DAB Solution B sequentially into 1 mL PBS, mix thoroughly to prepare the DAB chromogenic working solution.
Note: The prepared DAB color-developing solution must be used up completely at one time and should not be frozen for storage.
10. Color Development
1) Reaction: Add 100 μL of Streptavidin-HRP working solution onto the sample and incubate at room temperature for 30 minutes.
2) Washing: Discard the Streptavidin-HRP working solution and wash twice with PBS.
3) 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.
4) Termination: Discard the DAB color development solution and wash three times with PBS to terminate the color development reaction.
5) Cell nucleus staining (optional): Use hematoxylin staining solution or methyl green staining solution for cell nucleus staining.
6) Washing: Discard the cell nucleus staining solution and wash twice with PBS.
7) 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.
Notes
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.
Results Presentation
Figure 1. TUNEL staining for apoptotic cells. No obvious positive staining was observed in the untreated control group. Abundant brown TUNEL-positive nuclei with fragmented DNA were detected in the DNase I‑treated positive control group, which verified the performance of the TUNEL staining kit. Consistent with the induction of apoptosis, Staurosporine‑treated cells also exhibited prominent brown TUNEL‑positive nuclei, further confirming that this kit effectively detects apoptotic cell death triggered by chemical inducers. These results demonstrate the high sensitivity and reliability of the Colimeric TUNEL assay for evaluating apoptosis in various experimental models.
ZJ26F0939004
危险品化学品经营许可证（带存储）