计算溶液所需的质量、体积或浓度。
为什么选择此级别
，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
避光,-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
细胞发生凋亡时, 会激活一些DNA内切酶，这些内切酶会切断核小体间的基因组DNA，产生180 bp-200 bp的 DNA 片段，表现为琼脂糖凝胶电泳中呈现的特异的梯状Ladder图谱。基因组DNA双链或单链断裂时会出现产生大量的粘性3'-OH末端，可在脱氧核糖核苷酸末端转移酶（TdT）的催化作用下，与aladdin®/Cy-dUTP结合，从而通过荧光显微镜或流式细胞仪直接进行凋亡细胞的检测，这类方法称为脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（Terminal -deoxynucleotidyl transferase mediated nick end labeling, TUNEL）。由于正常的或正在增殖的细胞几乎没有DNA的断裂，因而没有3'-OH形成，很少能够被染色。Tunel法可以对完整的单个凋亡细胞核或凋亡小体进行原位染色，能准确地反应细胞凋亡典型的生物化学和形态特征，可检测出极少量的凋亡细胞，因而在细胞凋亡的研究中广泛采用。 本试剂盒应用范围广，可用于检测冷冻或石蜡切片中的细胞凋亡情况，也可检测培养的贴壁细胞或悬浮细胞的凋亡情况。可选择性的检测凋亡细胞，而非坏死细胞或因辐照和药物治疗而造成的 DNA 链断裂的细胞。本试剂盒检测细胞凋亡，耗时短，只需一步染色反应，洗涤后即可检测。
组分：
产品参数：
555/565 nm
使用方法：
实验材料（自备）
.PBS 缓冲液（1×, pH~7.4）
.0.2% Triton X- 100（PBS 配制）
.0.1% Triton X- 100（PBS 配制，其中含 5 mg/mLBSA）
.4%多聚甲醛（PBS 配制）
.免疫组化笔
.脱蜡溶剂（石蜡切片样本）
.石蜡切片处理相关试剂
.抗荧光淬灭封片剂
.ddH2O
实验设计
.A. 阳性对照：
DNaseI 处理制备阳性对照载玻片。 DNaseI 可以消化单链或双链 DNA 暴露 3'-OH 末端，人为造成细胞凋亡。 每次实验做一次即可。（用来验证本次实验操作和试剂盒有无问题）
.B. 阴性对照：
使用不含 TdT Enzyme 的 TUNEL Reaction Buffer，用 ddH2O 替代 TdT Enzyme 。 （主要是排除细胞自身凋亡、操作过程等原因导致的非特异性染色； 以及调整拍摄曝光强度。）
.C. 实验处理组。
实验组按照说明书正常操作。
.D. 实验对照组。
实验组按照说明书正常操作。
实验步骤
1. 样本准备：
（1）对于贴壁细胞或细胞涂片
a. PBS 清洗 1 次。
注：如果担心细胞涂片的细胞贴得不牢，可以干燥样品使细胞贴得更牢。
b. 固定： 加入适量 4%多聚甲醛（PBS 配制）， 4℃固定 30 min 。PBS 清洗 2 次。
c. 通透： 加入适量 0.2% Triton X- 100（PBS 配制），室温通透 20 min 。PBS 清洗 2 次。
d. 转步骤 2. TUNEL 反应。
（2）对于悬浮细胞或细胞悬液
a. 收集细胞（ 3-5×106 个细胞），1000 rpm 离心 5 min ，PBS 清洗 2 次。
b. 固定： 加入适量 4%多聚甲醛（PBS 配制）充分重悬细胞， 4℃固定 30 min 。2000 rpm 离心 5 min ，PBS 清洗 2 次。
c. 通透： 加入适量 0.2% Triton X- 100（PBS 配制），室温通透 20 min 。2000 rpm 离心 5 min ，PBS 清洗 2 次。
d. 转步骤 2. TUNEL 反应。
（3）石蜡组织切片
a. 脱蜡与水化： 将切片样本依次放入二甲苯 I（10 min）→ 二甲苯 II（10 min）→ 100％乙醇 I（5 min）→ 100％乙醇 II（5 min）→ 95%乙醇（5 min）→ 90%乙醇（5 min）→ 80%乙醇（5 min）→ 70%乙醇（5 min）→ ddH2O 冲洗 5 min ，冲洗 2次。
注：二甲苯有毒，易挥发，请在通风橱中进行此操作。
b. 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，以便下游通透与标记。
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。
c. 通透： 按 1: 100 的比例，将 2 mg/mL 的 ProteinaseK 溶液用 PBS 稀释至终浓度 20 µg/mL，在每个样本上滴加 100 µL，使溶液覆盖全部样本区域， 20-37℃孵育 20 min。
注： Proteinase K 可通透细胞膜和核膜，从而使后续步骤的染色试剂充分进入细胞核进行反应，提高标记效率。孵育时间过 长会增加组织切片在后续洗涤步骤中从载波片上脱落的风险， 过短则可能造成透性处理不充分， 影响标记效率。为得到更好的结果， Proteinase K 的浓度、孵育时间、温度需根据不同类型组织样本进行优化。
d. PBS 清洗切片 2 次，每次 5 min，用滤纸吸去多余的液体，将处理好的样本放在湿盒中保持湿润。
注：这一步必须把 Proteinase K 洗涤干净，否则会严重干扰后续的标记反应。
e. 转步骤 2. TUNEL 反应。
（4）冰冻组织切片
a. 固定： 取出冰冻切片，并回温至室温。加入适量 4%多聚甲醛（PBS 配制），室温固定 30 min 。PBS 清洗 2 次，每次 10min。
注： 若是担心甲醛清洗不干净， 影响最终染色效果。可在甲醛固定完成后加入适量 2 mg/mL 甘氨酸清洗 10 min，中和残留的固定液，再进行 PBS 清洗。
b. 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，以便下游通透与标记。
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。
c. 通透： 按 1: 100 的比例，将 2 mg/mL 的 ProteinaseK 溶液用 PBS 稀释至终浓度 20 µg/mL，在每个样本上滴加 100 µL，使溶液覆盖全部样本区域， 20-37℃孵育 20 min。
注： Proteinase K 可通透细胞膜和核膜，从而使后续步骤的染色试剂充分进入细胞核进行反应，提高标记效率。孵育时间过 长会增加组织切片在后续洗涤步骤中从载波片上脱落的风险， 过短则可能造成透性处理不充分， 影响标记效率。为得到更好的结果， Proteinase K 的浓度、孵育时间、温度需根据不同类型组织样本进行优化。
d. PBS 清洗切片 2 次，每次 5 min，用滤纸吸去多余的液体，将处理好的样本放在湿盒中保持湿润。
注：这一步必须把 Proteinase K 洗涤干净，否则会严重干扰后续的标记反应。
e. 转步骤 2. TUNEL 反应。
（5）阳性处理（仅阳性对照进行此步骤，其他样品直接进行 TUNEL 反应步骤）
a. 按 1: 10 的比例用 ddH2O 将 10×DNase I Buffer 稀释成 1×DNase I Buffer 备用。
b. 滴加 100 µL 1×DNase I Buffer 到已处理的样本上， 覆盖全部样本区域，室温平衡 5 min。
c. 用 1×DNase I Buffer 以 1: 100 稀释 DNase I (2 U/μL)至终浓度 20 U/mL 的工作液。
d. 弃去 Buffer，加入 100 μL 浓度为 20 U/mL 的 DNase I 工作液，室温孵育 10 min。
e. 弃去 DNase I 工作液， PBS 清洗 2 次。
f. 转步骤 2. TUNEL 反应。
2. TUNEL 反应
（1）配制 TUNEL 反应液（即用即配） ：
|
（2）对于贴壁细胞、细胞涂片或组织切片
a.每个样本加入 50 μL TUNEL 反应液，使反应液均匀覆盖样本。 37℃避光孵育适宜的时间（细胞推荐染色时间 30 min- 1 h，组织染色时间推荐 2 h）。
注： 50 μL TUNEL 反应液适合涂片、切片或 96 孔板（其他不同孔板可以适当调整 TUNEL 反应液体积，覆盖细胞即可） 。 如果待检测的样品为涂片、切片或在 24 孔板、 12 孔板或 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 TUNEL 反应液后覆盖在样品上，可以防止 TUNEL 反应液蒸发，并且使TUNEL 反应液均匀覆盖样本。
b. 弃去 TUNEL 反应液， PBS 清洗 2 次后， 再用 0.1% Triton X- 100（PBS 配制，其中含 5 mg/mL BSA）清洗 3 次，每次 5min，这样游离的未反应的标记物可以清除较干净。
c. （可选） 每个样本加入适量浓度为 5 μg/mL 的 DAPI 染液， 室温避光孵育 5 min。染色完成后， 弃去 DAPI 染液， PBS 清洗2 次，每次 5 min。
d. （可选） 切片封片：每个样本滴加 50 μL 抗荧光淬灭封片剂（抗荧光淬灭封片剂可能会不适用于某些染料， 实验前建议进行预实验测试匹配性），盖上盖玻片，用镊子的钝端轻轻敲击盖玻片，去除气泡以使封片完全。
e. 用滤纸吸去多余的液体，向样本区域加 100 μL PBS 保持样本湿润，立即在荧光显微镜下观察。
（3）对于悬浮细胞或细胞悬液
a.每个样本管加入 50 μLTUNEL 反应液轻轻重悬细胞，37℃避光孵育 30~ 1 h。每隔 15 min 用微量移液器轻轻重悬细胞。
b. 2000 rpm 离心 5 min，弃去 TUNEL 反应液， 用 0.1% Triton X- 100（PBS 配制，其中含 5 mg/mLBSA）清洗 2 次，每次 5min，这样游离的未反应的标记物可以清除较干净。
c. 每个样本管加入 100 μL 浓度为 5 μg/mL 的 DAPI 染液，室温避光孵育 5 min。
d. 加入 400 μL PBS 重悬细胞，立即用流式细胞仪检测或进行涂片后在荧光显微镜下观察。
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.染色背景较重或非特异性着色明显时可适当减少染色时间。
3.实验时建议增加阴性对照和阳性对照组。
4.组分A使用时请佩戴口罩、手套，如接触皮肤，请立即用大量水冲洗。
5.荧光染料均存在淬灭问题，请尽量注意避光，以减缓荧光淬灭。
6.为了您的安全和健康，请穿实验服并戴一次性手套操作。
应用范围：
晚期凋亡检测、TUNEL Kit
When apoptosis occurs, some DNA endonucleases will be activated. These endonucleases will cut off genomic DNA between nucleosomes and produce 180 bp-200 BP DNA fragments, which appear as a specific ladder pattern in agarose gel electrophoresis. When double strand or single strand breaks occur in genomic DNA, a large number of sticky 3'-oh ends will be generated, which can interact with YF under the catalysis of deoxyribonucleotide terminal transferase (TDT) ®/ CY dUTP binding can directly detect apoptotic cells by fluorescence microscopy or flow cytometry. This kind of method is called terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL). Because normal or proliferating cells have almost no DNA breaks, there is no 3'-oh formation and they can rarely be stained. TUNEL method can stain intact single apoptotic nuclei or apoptotic bodies in situ, can accurately reflect the typical biochemical and morphological characteristics of apoptosis, and can detect a very small number of apoptotic cells, so it is widely used in the study of apoptosis. This kit has a wide range of applications and can be used to detect apoptosis in frozen or paraffin sections, as well as cultured adherent cells or suspended cells. It can selectively detect apoptotic cells, but not necrotic cells or cells with DNA strand breaks caused by irradiation and drug treatment. This kit detects cell apoptosis with a short time-consuming, one-step staining reaction and can be detected after washing.
Product parameters：
555/565 nm
Component：
Instruction：
Experimental materials (self provided)
PBS buffer (1 x, pH~7.4)
. 0.2% Triton X -100 (PBS formulation)
. 0.1% Triton X -100 (PBS formulation, containing 5 mg/mLBSA)
4% paraformaldehyde (prepared with PBS)
Immunohistochemical pen
Dewaxing solvent (paraffin section sample)
Related reagents for paraffin section processing
Anti fluorescence quenching and sealing agent
. ddH2O
experimental design
. A. Positive control:
Prepare positive control slides using DNaseI treatment. DNaseI can digest single or double stranded DNA and expose the 3 '- OH end, artificially causing cell apoptosis. One experiment per time is sufficient. (To verify if there are any issues with the experimental operation and reagent kit)
B. Negative control:
Use TUNEL Reaction Buffer without TdT Enzyme and replace TdT Enzyme with ddH2O. (Mainly to exclude non-specific staining caused by cell apoptosis, operational processes, and other reasons; and to adjust the exposure intensity of the shooting.)
C. Experimental processing group.
The experimental group operated normally according to the instructions.
D. Experimental control group.
The experimental group operated normally according to the instructions.
Experimental steps
1. Sample preparation:
(1) For adherent cells or cell smears
a. Clean once with PBS.
Note: If you are concerned that the cells on the cell smear may not adhere firmly, you can dry the sample to make the cells adhere more firmly.
b. Fixation: Add an appropriate amount of 4% paraformaldehyde (prepared with PBS) and fix at 4 ℃ for 30 minutes. Clean twice with PBS.
c. Translucency: Add an appropriate amount of 0.2% Triton X -100 (prepared with PBS) and let it penetrate at room temperature for 20 minutes. Clean twice with PBS.
d. Step 2: TUNEL reaction.
(2) For suspended cells or cell suspensions
a. Collect cells (3-5 x 106 cells), centrifuge at 1000 rpm for 5 minutes, and wash twice with PBS.
b. Fixation: Add an appropriate amount of 4% paraformaldehyde (prepared with PBS) and resuspend the cells thoroughly. Fix at 4 ℃ for 30 minutes. Centrifuge at 2000 rpm for 5 minutes and clean twice with PBS.
c. Translucency: Add an appropriate amount of 0.2% Triton X -100 (prepared with PBS) and let it penetrate at room temperature for 20 minutes. Centrifuge at 2000 rpm for 5 minutes and clean twice with PBS.
d. Step 2: TUNEL reaction.
(3) Paraffin tissue sectioning
a. Dewaxing and hydration: Place the sliced samples sequentially in xylene I (10 min) → xylene II (10 min) → 100% ethanol I (5 min) → 100% ethanol II (5 min) → 95% ethanol (5 min) → 90% ethanol (5 min) → 80% ethanol (5 min) → 70% ethanol (5 min) → ddH2O rinse for 5 min, rinse twice.
Note: Xylene is toxic and volatile. Please perform this operation in a fume hood.
b. Use filter paper to dry the liquid around the sliced sample, and circle the sample contour with an immunohistochemical pen for downstream transparency and labeling.
Note: If it is found that the contour circle of immunohistochemistry strokes is damaged in subsequent experimental operations, it needs to be redrawn in a timely manner.
c. Transparency: Dilute 2 mg/mL of ProteinaseK solution with PBS in a ratio of 1:100 to a final concentration of 20 µ g/mL. Add 100 µ L dropwise to each sample to cover all sample areas. Incubate at 20-37 ℃ for 20 minutes.
Note: Protein K can penetrate the cell membrane and nuclear membrane, allowing subsequent staining reagents to fully enter the nucleus for reaction and improve labeling efficiency. An excessively long incubation time increases the risk of tissue slices falling off the carrier film during subsequent washing steps, while a too short incubation time may result in insufficient permeability treatment and affect labeling efficiency. To obtain better results, the concentration, incubation time, and temperature of Protein K need to be optimized according to different types of tissue samples.
d. Wash the slices twice with PBS, each time for 5 minutes. Use filter paper to remove excess liquid, and place the processed sample in a wet box to keep it moist.
Note: Protein K must be washed thoroughly in this step, otherwise it will seriously interfere with subsequent labeling reactions.
e. Step 2: TUNEL reaction.
(4) Frozen tissue sections
a. Fixation: Take out frozen sections and warm them back to room temperature. Add an appropriate amount of 4% paraformaldehyde (prepared with PBS) and fix at room temperature for 30 minutes. Wash twice with PBS for 10 minutes each time.
Note: If you are concerned that formaldehyde cleaning may not be clean enough, it may affect the final dyeing effect. After formaldehyde fixation is completed, an appropriate amount of 2 mg/mL glycine can be added and washed for 10 minutes to neutralize the residual fixing solution, and then PBS cleaning can be carried out.
b. Use filter paper to dry the liquid around the sliced sample, and circle the sample contour with an immunohistochemical pen for downstream transparency and labeling.
Note: If it is found that the contour circle of immunohistochemistry strokes is damaged in subsequent experimental operations, it needs to be redrawn in a timely manner.
c. Transparency: Dilute 2 mg/mL of ProteinaseK solution with PBS in a ratio of 1:100 to a final concentration of 20 µ g/mL. Add 100 µ L dropwise to each sample to cover all sample areas. Incubate at 20-37 ℃ for 20 minutes.
Note: Protein K can penetrate the cell membrane and nuclear membrane, allowing subsequent staining reagents to fully enter the nucleus for reaction and improve labeling efficiency. An excessively long incubation time increases the risk of tissue slices falling off the carrier film during subsequent washing steps, while a too short incubation time may result in insufficient permeability treatment and affect labeling efficiency. To obtain better results, the concentration, incubation time, and temperature of Protein K need to be optimized according to different types of tissue samples.
d. Wash the slices twice with PBS, each time for 5 minutes. Use filter paper to remove excess liquid, and place the processed sample in a wet box to keep it moist.
Note: Protein K must be washed thoroughly in this step, otherwise it will seriously interfere with subsequent labeling reactions.
e. Step 2: TUNEL reaction.
(5) Positive treatment (only the positive control is subjected to this step, and other samples are directly subjected to the TUNEL reaction step)
a. Dilute 10 x DNase I Buffer with ddH2O in a ratio of 1:10 to 1 x DNase I Buffer for later use.
b. Drip 100 µ L of 1xDNase I Buffer onto the processed sample, covering all sample areas, and equilibrate at room temperature for 5 minutes.
c. Dilute DNase I (2 U) with 1 x DNase I Buffer at a ratio of 1:100/ μ L) A working solution with a final concentration of 20 U/mL.
d. Discard the buffer and add 100 μ Incubate DNase I working solution with a concentration of 20 U/mL at room temperature for 10 minutes.
e. Discard DNase I working solution and clean twice with PBS.
f. Step 2: TUNEL reaction.
2. TUNEL reaction
(1) Prepare TUNEL reaction solution (ready to use):
|
(2) For adherent cells, cell smears, or tissue sections
a. Add 50 to each sample μ L TUNEL reaction solution, evenly cover the sample with the reaction solution. The appropriate time for dark incubation at 37 ℃ (recommended staining time for cells is 30 minutes to 1 hour, and tissue staining time is 2 hours).
Note: 50 μ L TUNEL reaction solution is suitable for smear, slicing, or 96 well plates (other different well plates can adjust the volume of TUNEL reaction solution appropriately to cover cells). If the sample to be tested is a smear, slice, or in a 24 well plate, 12 well plate, or 6 well plate, anti evaporation film can be used, or self sealing bags or other appropriate materials can be used to cut circular plastic sheets slightly smaller than the holes. After adding TUNEL reaction solution dropwise, cover the sample to prevent the evaporation of TUNEL reaction solution and make the TUNEL reaction solution evenly cover the sample.
b. Discard the TUNEL reaction solution, wash twice with PBS, and then wash three times with 0.1% Triton X -100 (PBS preparation, containing 5 mg/mL BSA) for 5 minutes each time. This way, free unreacted markers can be removed cleanly.
c. (Optional) Add an appropriate concentration of 5 to each sample μ DAPI staining solution with a concentration of g/mL, incubated at room temperature in dark for 5 minutes. After staining, discard DAPI staining solution and wash twice with PBS for 5 minutes each time.
d. (Optional) Slice sealing: Add 50 drops to each sample μ L anti fluorescence quenching sealing agent (anti fluorescence quenching sealing agent may not be suitable for certain dyes, it is recommended to conduct pre experimental testing for compatibility before the experiment), cover the cover glass, gently tap the cover glass with the blunt end of tweezers to remove bubbles and ensure complete sealing.
e. Use filter paper to remove excess liquid and add 100 to the sample area μ Keep the sample moist with PBS and immediately observe under a fluorescence microscope.
(3) For suspended cells or cell suspensions
a. Add 50 to each sample tube μ Gently resuspend cells in LTUNEL reaction solution and incubate at 37 ℃ in the dark for 30-1 hour. Gently resuspend cells with a micropipette every 15 minutes.
b. Centrifuge at 2000 rpm for 5 minutes, discard TUNEL reaction solution, and wash twice with 0.1% Triton X -100 (PBS preparation, containing 5 mg/mLBSA) for 5 minutes each time. This way, free unreacted markers can be removed cleanly.
c. Add 100 to each sample tube μ L concentration is 5 μ DAPI staining solution with a concentration of g/mL, incubated at room temperature in dark for 5 minutes.
d. Join 400 μ L PBS resuspended cells and immediately detected with a flow cytometer or observed under a fluorescence microscope after smearing.
Matters needing attention：
1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments.
2. when the staining background is heavy or non-specific staining is obvious, the staining time can be appropriately reduced.
3. it is recommended to add negative control and positive control groups during the experiment.
4. please wear mask and gloves when using component A. if it contacts the skin, please wash it with plenty of water immediately.
5. fluorescent dyes have quenching problems. Please try to avoid light to slow down fluorescence quenching.
6. for your safety and health, please wear experimental clothes and disposable gloves.
Scope of application：
Late apoptosis detection, TUNEL Kit
规格
英文名称
Aladdin ® 555 TUNEL apoptosis Kit (orange red fluorescence)
备注
组分A需避光，避免反复冻融
储存与运输
储存条件
避光,-20°C储存
运输条件
超低温运输
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
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