本试剂盒采用高灵敏度的TUNEL（TdT-mediated dUTP Nick End Labeling）法，旨在简单、快速地检测细胞凋亡过程中发生的DNA片段化。对于经过固定和洗涤的细胞或组织，只要经过一步染色反应，洗涤后就可以通过荧光显微镜或流式细胞仪进行悬浮细胞、贴壁细胞的流式凋亡检测。细胞凋亡时，内源性核酸内切酶被激活，将基因组DNA切割成180-200bp的片段，暴露出大量3‘-OH末端。在末端脱氧核糖核苷酸转移酶（TdT）的催化下，荧光素（如Cy3或AF555）标记的dUTP会掺入到这些断裂位点，从而通过荧光显微镜或流式细胞仪检测到凋亡细胞发出的红色荧光。

本试剂盒适用于多种样本类型，包括石蜡切片、冰冻切片、细胞爬片、细胞涂片及悬浮细胞，无需额外使用二抗，大大简化了实验流程。

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

O1524963	组件	物理外观	20T	50T	储存温度	单次测试使用量
O1524963A	TdT酶	液体	20 μL	50 μL	-20℃.	1 μL
O1524963B	荧光标记液	液体	40 μL	100 μL	-20℃. 避光	2 μL
O1524963C	反应缓冲液 (5×)	液体	200 μL	500 μL	-20℃.	10 μL
O1524963D	星形孢菌素 (5mM)	液体	20 μL	50μL	-20℃.	As needed
O1524963E	DNase I (10U/μL)	液体	20 μL	50μL	-20℃.	As needed

使用方法

1. 贴壁细胞 (爬片/涂片

1) 将细胞爬片放入细胞培养板中，以2×10⁶ cells/mL的浓度将细胞重悬于1×PBS 中。吸取50-100 µL细胞悬液放入细胞培养板中，接种细胞进行培养，至少24 h。

2) 固定：吸弃培养基，PBS洗2次。加入4%多聚甲醛室温固定15-30分钟。

3) 洗涤：PBS漂洗2次。

4) 通透：加入0.3% Triton X-100室温孵育5-10分钟。

5) 洗涤：PBS漂洗2-3次，用吸水纸吸干样本周围水分(请勿干燥)。

6) 转步骤5。

2. 悬浮细胞

1) 收集：离心收集细胞并进行计数，每组取1×10⁶ 个细胞，300g 离心5 min，弃上清。

2) 固定：加入4%多聚甲醛，4°C固定15-30分钟(建议摇床缓慢摇晃以防细胞聚集)。

3) 洗涤：离心(300g，5分钟)，弃上清，PBS重悬洗涤1次。

4) 通透：加入0.3% Triton X-100室温孵育5-10分钟。

5) 洗涤：离心，PBS洗涤2次终止破膜，洗涤后收集细胞沉淀。

6) 转步骤5。

3. 石蜡切片

1) 脱蜡水化：二甲苯浸泡2次，每次10分钟，然后用无水乙醇2次，每次5分钟，95%，85%，75%梯度乙醇各洗涤1次，每次3分钟，最后用PBS浸泡5分钟。

2) 通透：滴加Proteinase K工作液(P274341，20 μg/mL，用PBS稀释)，37°C孵育15-30分钟。

3) 洗涤：PBS漂洗3次。

4) 转步骤5。

4. 冰冻切片

1) 复温固定：切片恢复室温，浸入4%多聚甲醛室温固定15-30分钟。

2) 洗涤：PBS漂洗2次。

3) 通透：滴加Proteinase K工作液，室温孵育10-20分钟。

4) 洗涤：PBS漂洗3次。

注意：预处理后的样本建议放在湿盒中保持湿润，防止干片导致高背景。

5) 转步骤5。

5. 样本平衡

将反应缓冲液(5×)用去离子水稀释为反应缓冲液(1×)，加入50 μL反应缓冲液(1×)，轻轻吹打混匀，将样本至于37°C培养箱平衡15min。

6. TUNEL反应液配制及标记

1）参考以下比例配制反应液，配制时须在冰上避光操作并充分混合均匀，每个样本 (50 μL体系) 所需体积计算：

反应组分	每孔体积（μL）
TdT酶	1
荧光标记液	2
反应缓冲液(5×)	10
去离子水	37
总体积	50

注：50 μL TUNEL反应液适合涂片、切片或96孔板、48孔板、24孔板或12孔板或12孔板的一个孔，如果是6孔板中，可以使用防蒸发膜，或自行尝试使用自封袋或者其它适当材料自行裁剪成比孔略小的圆形塑料片，滴加6孔板中的一个孔TUNEL检测液宜使用100 μL。

2）阴性对照：用ddH₂O替代TdT Enzyme；阳性对照：向通透后的样本(如果是悬浮细胞，需要离心去除上清)，加入DNase I工作液(将DNase I 10U/μL用PBS稀释至100 U/mL)室温孵育10分钟；或者用5 μM的星形孢菌素刺激过夜，用PBS洗净后再进行TUNEL标记。

注：阳性对照组必须与其它实验组分开处理，否则残留的 DNase I 可能会使实验组出现假阳性信号。

3）标记反应：吸尽样本表面的多余液体。滴加 50 μL TUNEL反应液，确保完全覆盖样本。将载玻片置于湿盒中，37°C 避光孵育 60分钟。洗涤：PBS漂洗3次，每次5分钟。

7. 荧光显微镜分析

1）核复染：滴加DAPI(D1372407，1 μg/mL)，室温避光孵育5分钟。

2）洗涤：PBS漂洗3次。

3）封片：滴加抗荧光淬灭封片剂，盖上盖玻片。

4）镜检：在荧光显微下观察分析样本。Cy3红色荧光染料的最大激发波长是550 nm，最大发射波长是570 nm；DAPI与双链DNA结合时最大激发波长是358 nm，最大发射波长是461 nm。

8. 流式细胞仪分析

1) 300g 离心细胞。除去上清液，重悬于1 mL PBS 中。

2) 重复洗涤2次。

3) 重悬于200 µL DAPI(1 μg/mL)，室温避光孵育5分钟。

4) 通过流式细胞仪分析细胞。参考上述激发/发射波长。

注意事项

1、避光操作：从加入标记液开始，后续所有步骤均需严格避光，以防荧光淬灭。

2、防止干片：在滴加TUNEL反应液后，若样本体积较小，建议使用湿盒或封口膜覆盖，防止试剂蒸发导致假阴性。

3、阳性对照组需隔离：DNase I处理阳性对照时，必须使用单独的染色缸或离心管，避免DNase I残留污染实验组，导致假阳性。

4、蛋白酶K优化：Proteinase K处理时间过长易导致切片脱落或细胞形态破坏，过短则通透不充分。建议预实验摸索最佳时间。

5、自发荧光：若组织（如肝脏、肾脏）或固定液导致背景荧光较强，可在通透后使用自发荧光淬灭剂处理。

6、安全操作：二甲苯、多聚甲醛及TdT酶均为有害物质，操作时请穿实验服，戴手套。

结果展示

图1. TUNEL荧光染色检测Jurkat细胞凋亡结果。DAPI 蓝色荧光标记全部细胞核，Cy3 红色荧光标记TUNEL阳性凋亡细胞；对照组几乎无红色凋亡荧光，星形孢菌素(Staurosporine)处理组可见大量 Cy3 阳性凋亡细胞。

图2. 流式细胞术检测Jurkat细胞凋亡。蓝色曲线为未刺激对照组，绿色曲线为5 μM 星形孢菌素处理过夜；虚线：未进行TUNEL标记的细胞，实线：经TUNEL试剂盒标记检测荧光的细胞。

This kit employs a highly sensitive TUNEL (TdT-mediated dUTP Nick End Labeling) method, designed for simple and rapid detection of DNA fragmentation occurring during apoptosis. For fixed and washed cells or tissues, after just a one-step staining reaction and subsequent washing, apoptotic cells can be detected by fluorescence microscopy or flow cytometry for both suspension and adherent cells. During apoptosis, endogenous endonucleases are activated, cleaving genomic DNA into fragments of 180-200 bp and exposing a large number of 3'-OH ends. Under the catalysis of terminal deoxynucleotidyl transferase (TdT), fluorophore-labeled dUTP (such as Cy3, AF555) is incorporated into these break sites, allowing apoptotic cells to be detected by red fluorescence using a fluorescence microscope or flow cytometer.

This kit is suitable for a wide range of sample types, including paraffin-embedded sections, frozen sections, cell smears, cytospin preparations, and suspension cells. It does not require the use of secondary antibodies, greatly simplifying the experimental procedure.

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

O1524963	Components	Appearance	20T	50T	Storage	Quantity Per Test
O1524963A	Recombinant TdT Enzyme	Liquid	20 μL	50 μL	-20℃.	1 μL
O1524963B	Cy3-dUTP Labeling Solution	Liquid	40 μL	100 μL	-20℃. Store in the dark	2 μL
O1524963C	Reaction Buffer (5×)	Liquid	200 μL	500 μL	-20℃.	10 μL
O1524963D	Staurosporine (5mM)	Liquid	20 μL	50 μL	-20℃.	As needed
O1524963E	DNase I(10U/μL)	Liquid	20 μL	50 μL	-20℃.	As needed

Protocol

1. Adherent Cells (Coverslip Culture / Smear Preparation)

1) Place the coverslips into the wells of the cell culture plate. Resuspend the cells in 1× PBS at a concentration of 2×10⁶ cells/mL. Transfer 50-100 µL of the cell suspension into each well of the plate, and culture the cells for at least 24 h to allow for cell attachment.

2) Fixation: Aspirate culture medium and rinse twice with PBS. Add 4% paraformaldehyde and incubate at room temperature for 15-30 min for fixation.

3) Washing: Rinse twice with PBS.

4) Permeabilization: Add 0.3% Triton X-100 and incubate at room temperature for 5-10 min.

5) Washing: Rinse 2-3 times with PBS; blot residual liquid around specimens with filter paper (avoid complete desiccation of samples).

6) Continue to Step 5.

2. Suspension Cells

1) Collect the cells by centrifugation and determine the cell number. Aliquot 1×10⁶ cells per group, centrifuge at 300 g for 5 min, and discard the supernatant.

2) Fixation: Add 4% paraformaldehyde, fix at 4 °C for 15-30 min (gentle shaking on a rocker is recommended to prevent cell clumping).

3) Washing: Centrifuge at 300 g for 5 min, discard supernatant, resuspend pellet in PBS for one round of washing.

4) Permeabilization: Add 0.3% Triton X-100 and incubate at room temperature for 5-10 min.

5) Washing: Centrifuge the cells, wash twice with PBS to terminate permeabilization, and collect the cell pellet after washing.

6) Continue to Step 5.

3. Paraffin-Embedded Tissue Sections

1) Deparaffinization and hydration: Immerse sections in xylene twice (10 min per immersion); sequentially dehydrate in absolute ethanol (2×5 min), 95% ethanol, 85% ethanol and 75% ethanol (3 min for each concentration); equilibrate in PBS for 5 min at last.

2) Permeabilization: Drop Proteinase K working solution (P274341, 20 μg/mL, diluted with PBS) onto sections and incubate at 37 °C for 15-30 min.

3) Washing: Rinse three times with PBS.

4) Continue to Step 5.

4. Frozen Tissue Sections

1) Rewarming & fixation: Leave sections to equilibrate to ambient temperature, fix in 4% paraformaldehyde at room temperature for 15-30 min.

2) Washing: Rinse twice with PBS.

3) Permeabilization: Apply Proteinase K working solution and incubate at room temperature for 10-20 min.

4) Washing: Rinse three times with PBS.

5) Continue to Step 5.

5. Sample Equilibration

Dilute the 5× Reaction Buffer with deionized water to prepare 1× Reaction Buffer. Add 50 μL of the 1× Reaction Buffer to the sample, mix gently by pipetting, and incubate the sample in a 37°C incubator for 15 min to allow equilibration.

6、Preparation of TUNEL Reaction Solution and Labeling

1）Prepare reaction solution according to the ratio below. Carry out preparation on ice under dark conditions and mix thoroughly. Calculate required volume per sample (50 μL final system):

Components of TUNEL Reaction Solution	Volume per well (μL)
TdT Enzyme	1
Cy3-dUTP Labeling Solution	2
Reaction Buffer(5×)	10
Deionized Water	37
Total Volume	50

Note: 50 μL of TUNEL Detection Solution is suitable for use with smears, tissue sections, or one well of a 96-well, 48-well, 24-well, or 12-well plate. For use in a 6-well plate, an anti-evaporation film may be employed. Alternatively, a plastic sheet (e.g., cut from a resealable bag or other suitable material) may be trimmed into a round piece slightly smaller than the well diameter and placed over the sample to prevent evaporation. When processing one well of a 6-well plate, it is recommended to use 100 μL of TUNEL Detection Solution.

2）Negative control: Replace TdT Enzyme with ddH₂O. Positive Control: After permeabilization, add DNase I working solution (Dilute DNase I (10 U/μL) with PBS to a final concentration of 100 U/mL.) to the samples and incubate for 10 minutes at room temperature. For suspension cells, remove the supernatant by centrifugation prior to the addition of DNase I. Alternatively, treat the cells with 5 µM staurosporine overnight, then wash thoroughly with PBS before proceeding with the TUNEL labeling.

3）Labeling reaction: Aspirate excess liquid on sample surface. Add 50 μL TUNEL working solution to fully cover the specimen. Place slides in a humidified chamber and incubate at 37 °C in the dark for 60 min. Washing step: Rinse 3 times with PBS, 5 min per wash.

7、Counterstaining, Mounting and Microscopic Observation

1）Nuclear counterstaining: Add DAPI (D1372407, 1 μg/mL) solution, incubate at room temperature in the dark for 5 min.

2）Washing: Rinse three times with PBS.

3）Mounting: Drop anti-fade mounting medium and place coverslips.

4）Microscopic Examination: Observe and analyze the samples under a fluorescence microscope. Cy3 red fluorescent dye has a maximum excitation wavelength of 550 nm and a maximum emission wavelength of 570 nm. DAPI, when bound to double-stranded DNA, exhibits a maximum excitation wavelength of 358 nm and a maximum emission wavelength of 461 nm.

8. Flow Cytometric Analysis

1) Centrifuge the cells at 300 g. Discard the supernatant and resuspend the cells in 1 mL of PBS.

2) Wash twice by repeating the centrifugation and resuspension steps.

3) Resuspend the cells in 200 µL of DAPI (1 µg/mL), and incubate for 5 minutes at room temperature in the dark.

4) Analyze the cells using a flow cytometer. Refer to the excitation/emission wavelengths mentioned above.

Notes

1. Protect from light: From the step of adding the labeling mix onward, all subsequent steps must be strictly performed in the dark (avoid light exposure) to prevent fluorescence quenching.

2. Prevent drying of samples: After adding the TUNEL reaction solution, if the sample volume is small, it is recommended to use a humidified chamber or cover with Parafilm to prevent evaporation of the reagent, which may lead to false-negative results.

3. Isolate positive controls: When treating positive controls with DNase I, a separate staining jar or centrifuge tube must be used to avoid DNase I carryover contaminating the experimental samples, which may cause false-positive results.

4. Optimize Proteinase K treatment: Excessive Proteinase K treatment time can easily lead to section detachment or disruption of cell morphology, while insufficient treatment results in inadequate permeabilization. It is recommended to perform preliminary experiments to determine the optimal incubation time.

5. Autofluorescence: If tissues (e.g., liver, kidney) or the fixative solution cause strong background fluorescence, treat the samples with an autofluorescence quencher after permeabilization.

6. Safety precautions: Xylene, paraformaldehyde, and TdT enzyme are all hazardous substances. Please wear a lab coat and gloves during handling.

Results Presentation

Figure 1. Detection of apoptosis in Jurkat cells via TUNEL fluorescence staining. All cell nuclei were counterstained with DAPI (blue), and TUNEL-positive apoptotic cells were labeled with Cy3 (red). Few red apoptotic signals were observed in the control group, whereas abundant Cy3-positive apoptotic cells were detected in the staurosporine-treated group.

Figure 2. Flow cytometric analysis of apoptosis in Jurkat cells. Blue curve represents the untreated control group, and green curve corresponds to cells treated overnight with 5 μM staurosporine. Dashed lines: cells without TUNEL labeling; solid lines: cells stained with TUNEL kit for fluorescence detection.

规格

规格或纯度

BioReagent, 适用于荧光分析, 生物技术级

英文名称

One-Step TUNEL Apoptosis Detection Kit (Red, with Apoptosis Positive Inducer)

应用

细胞凋亡

样品类型

细胞

检测方法

Flow Cytometry；IF

检测仪器

流式细胞仪,荧光显微镜

检测波长

Ex/Em=550/570nm

敏感性

Light-sensitive；Temperature-sensitive

Emission(nm)

570nm

Excitation(nm)

550nm

Ex/Em(nm)

Ex:550nm;Em:570nm

储存与运输

储存条件

-20°C储存

运输条件

超低温运输

稳定性与储存

各组分在相应的储存条件下保质期1年。

组分与储存

O1524963	组件	物理外观	20T	50T	储存温度	单次测试使用量
O1524963A	TdT酶	液体	20 μL	50 μL	-20℃.	1 μL
O1524963B	荧光标记液	液体	40 μL	100 μL	-20℃. 避光	2 μL
O1524963C	反应缓冲液 (5×)	液体	200 μL	500 μL	-20℃.	10 μL
O1524963D	星形孢菌素 (5mM)	液体	20 μL	50 μL	-20℃.	As needed
O1524963E	DNase I (10U/μL)	液体	20 μL	50 μL	-20℃.	As needed

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找到2个结果

批号(Lot Number)	证书类型	货号
ZJ26F0939014	分析证书	O1524963
ZJ26F0939013	分析证书	O1524963

技术文档和文章

o1524963 一步法tunel细胞凋亡检测试剂盒红色含凋亡阳性诱导剂 v2.pdf

o1524963 one-step tunel apoptosis detection kit red with apoptosis positive inducer.pdf

溶液计算器

摩尔浓度计算器

计算溶液所需的质量、体积或浓度。

质量

浓度

体积

分子量

g/mol

稀释计算器

计算配制储备溶液所需的稀释度。

浓度1（C1）

体积 1 (V1)

浓度 2 (C2)

体积 2 (V2)

复溶计算器

复溶计算器允许您快速计算试剂的体积，以复溶小瓶。只需输入试剂的质量和目标浓度，计算器将确定其余部分。

体积（添加到小瓶中）

质量（小瓶）

所需的复溶浓度

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一步法TUNEL细胞凋亡检测试剂盒 (红色, 含凋亡阳性诱导剂)