产品描述

细胞在发生凋亡时，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。细胞凋亡时抽提DNA进行电泳检测，可以发现180-200 bp的DNA ladder。 

TUNEL (TdT mediated dUTP Nick End Labeling)细胞凋亡检测试剂盒（FITC）可以用来检测组织细胞在凋亡晚期过程中细胞核DNA的断裂情况。其原理是在末端脱氧核糖核苷酸转移酶（Terminal Deoxynucleotidyl Transferase, TdT）的作用下，在基因组DNA断裂时暴露出的3´-羟基（3´-OH）末端掺入FITC-12-dUTP，从而可以用荧光显微镜或流式细胞仪检测。

本试剂盒对标记反应进行了优化，采用最佳比例的FITC-12-dUTP和未标记dNTP进行3’-OH末端的核苷酸掺入，使得同一个断裂的DNA片段末端可以形成更长的“标记尾巴”。该“标记尾巴”减少了相邻掺入dNTP上标记基团的空间位阻，增加每个断裂片段上的荧光基团数目，降低荧光基团相邻后可能造成的聚集和淬灭，从而提高检测灵敏度，减少非特异性反应。

本试剂盒应用范围广，可以用于检测冷冻或石蜡切片中的细胞凋亡情况，也可以检测培养的贴壁细胞或悬浮细胞的凋亡情况。

产品组分

运输与保存方法

冰袋（wet ice）运输。

本试剂盒储存在-20℃，FITC-12-dUTP Labling Mix避光储存于-20℃，保质期为二年。

注意事项

1）需自备用于洗涤细胞的PBS，用于封片的抗荧光淬灭封片液，用于固定的4%多聚甲醛。

2）如需染核，需自备DAPI（2 μg/mL）或PI（1 μg/mL）。

3）如果用流式细胞仪，自备PI（1 μg/mL）和DNase Free RNase A。

4）为了您的安全和健康，请穿实验服并戴一次性手套操作。

5）本产品仅作科研用途！

操作步骤

一、样品准备

A. 石蜡包埋组织切片

1. 室温下将石蜡组织切片放入二甲苯中浸泡5 min，重复一次，以彻底脱掉石蜡。

2. 室温下用100%乙醇浸泡切片5 min，重复一次。

3. 室温下用梯度乙醇（90、80、70%）各浸洗1次，每次3 min。

4. 用PBS轻轻润洗切片，并用滤纸小心吸干玻片上样本周围多余的液体。这时，可用石蜡笔或疏水笔在样品周围描绘样品分布的轮廓，便于下游透性处理和平衡标记操作。在实验过程中，切勿让样品干燥，处理好的样本放在湿盒中保持样本的湿润。

5. 配制Proteinase K工作液：按1:100的比例，用PBS作为稀释液来稀释2 mg/mL的Proteinase K溶液，使其终浓度为20 μg/mL。

6. 每个样本上滴加100 μL上述Proteinase K工作液，使其被全部覆盖，室温孵育20 min。

注：Proteinase K帮助组织和细胞对后续步骤的染色试剂通透。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。未得到更好的结果，可能需要优化Proteinase K孵育的时间。

7. 用PBS溶液润洗样本，轻轻去掉多余液体，并用滤纸小心吸干载玻片上样本周围的液体。处理后的样本放在湿盒中保存样本的湿润。

B. 组织冰冻切片

1. 将玻片浸没在4%多聚甲醛溶液（溶于PBS）中固定，室温下孵育15 min。

2. 轻轻去掉多余液体，并用滤纸小心吸干玻片上样本周围多余的液体。

3. 将玻片浸没在PBS溶液中，室温孵育15 min。

4. 轻轻去掉多余液体，并用滤纸小心吸干玻片上样本周围多余的液体。这时，可用石蜡笔或疏水笔在样品周围描绘样品分布的轮廓，便于下游透性处理和平衡标记操作。在实验过程中，切勿让样品干燥，处理好的样本放在湿盒中保持样本的湿润。

5. 配制Proteinase K工作液：按1:100的比例，用PBS作为稀释液来稀释2 mg/mL的Proteinase K溶液，使其终浓度为20 μg/mL。

6. 每个样本上滴加100 μL上述Proteinase K工作液，使其被全部覆盖，室温孵育10 min。

【注】Proteinase K帮助组织和细胞对后续步骤的染色试剂通透。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。未得到更好的结果，可能需要优化Proteinase K孵育的时间。

7. 用PBS溶液润洗样本2-3次。

8. 轻轻去掉多余液体，并用滤纸小心吸干载玻片上样本周围的液体。处理后的样本放在湿盒中保存样本的湿润。

C. 细胞样品

【细胞爬片的准备】

在Lab-Tek载玻片小室（Chamber Slides）上培养贴壁细胞。在凋亡诱导处理之后，用PBS洗2遍载玻片。

【细胞涂片的制备（以多聚赖氨酸包被的载玻片为例）】

1. 准备多聚赖氨酸包被的载玻片：吸取50–100 μL 0.01% (w/v)多聚赖氨酸水溶液，滴至每一片预清洗过的玻璃载玻片的表面。在将要用于固定细胞的区域将多聚赖氨酸溶液涂散为一薄层。待载玻片晾干之后，迅速用去离子水漂洗，然后让包被后的载玻片在空气中晾干30-60 min。包被后的载玻片能在室温储存数月。

2. 以约2×10⁷个细胞/mL的浓度将细胞重悬于PBS中，吸取50-100 μL细胞悬液滴于多聚赖氨酸包被的载玻片上，用一片干净的载玻片轻柔的涂开细胞悬液。

按照以下步骤对细胞样品进行处理：

1. 固定细胞，将载玻片浸入装有4%新鲜配制于PBS中的多聚甲醛的染色缸中，在4℃放置25 min。

2. 洗涤载玻片，将其浸入PBS中，室温放置5 min。重复用PBS洗一次。

3. 轻轻去掉多余液体，并用滤纸小心吸干玻片上样本周围多余的液体。这时，可用石蜡笔或指甲油在样品周围描绘样品分布的轮廓，便于下游透性处理和平衡标记操作。在实验过程中，切勿让样品干燥，处理好的样本放在湿盒中保持样本的湿润。

4. 每个样本上可浸于0.2%配制于PBS中的Triton X-100溶液中，室温孵育5 min进行通透处理（Proteinase K处理容易使细胞脱落）。

5. 在盛有PBS溶液的敞口烧杯中浸没清洗样本2-3次。

6. 轻轻去掉多余液体，并用滤纸小心吸干载玻片上样本周围的液体。处理后的样本放在湿盒中保存样本的湿润。

二、DNA酶处理阳性对照的步骤（可选）

在样本通透处理后，用DNA酶I处理细胞来准备阳性对照载玻片。该流程通常会引起被处理的大多数细胞显现绿色荧光。

【注】：DNA酶I处理固定的细胞会引起染色体DNA的断裂，产生许多可标记的DNA 3’-末端。

1. 按1:10的比例用去离子水稀释10×DNase I Buffer(每个样本需用200 μL 1×DNase I Buffer，即需要用20 μL 10×DNase I Buffer和180 μL去离子水混合稀释)，取其中100 μL滴加到已通透的样本上，室温孵育5 min。 向剩余100 μL 1×DNase I Buffer中加1 μL DNase I (1U/μL)，使其终浓度为10 U/mL。轻叩掉液体，加入100 μL含5.5-10 units/mL DNase I的缓冲液，室温孵育10 min。

2. 轻轻叩掉液体，加入100 μL 含10 U/mL DNase I 的缓冲液，室温孵育10 min。

3. 轻叩载玻片，去掉多余的液体，并将载玻片在装有去离子水的染色缸中彻底洗3-4次。

【注】：阳性对照载玻片必须使用单独的染色缸，否则阳性对照载玻片上残余的DNase I 可能会在实验载玻片上引入高背景。

三、标记与检测

1. 按1:5的比例用去离子水稀释5×Equilibration Buffer。

2. 每个样本滴加100 μL 1×Equilibration Buffer使其全部覆盖待检样本区域，室温孵育10-30 min。或者将载玻片放入一个含有 1×Equilibration Buffer的缸中，保证缓冲液没过样本。在平衡细胞的同时在冰上解冻FITC-12-dUTP Labling Mix，并且依照表1，准备足够量的用于所有实验的和可选阳性对照反应的TdT孵育缓冲液。对于面积小于5 cm²的一个标准反应，其体积是50 μL，用50 μL乘以实验和阳性对照反应的数目来确定所需TdT孵育缓冲液的总体积。对于表面积更大的样本，可成比例的增大试剂体积。

表1. 准备用于实验的和可选阳性对照反应的TdT孵育缓冲液

阴性对照体系：准备一份不含TdT酶的对照孵育缓冲液，用ddH₂O替代TdT酶。

3. 在平衡后的区域周围用吸水纸洗掉100 μL 1×Equilibration Buffer中的大部分，然后在5 cm²面积的细胞上加入50 μL TdT孵育缓冲液。不要让细胞干掉。这之后的操作，载玻片要避光。

4. 把塑料盖玻片盖在细胞上以保证试剂的平均分布，在湿盒的底部放上用水浸湿的纸巾。将载玻片置于湿盒内，在37℃孵育60 min。将湿盒用铝箔纸包裹以避光。

注：塑料盖玻片在使用前可以切成两半。折起盖玻片的边缘以便于移除和操作。

5. 移除塑料盖玻片，并将切片置于PBS溶液中室温孵育5 min。

6. 轻轻去掉多余液体，换用新鲜的PBS溶液室温孵育5 min，重复一次。

7. 用滤纸轻轻擦掉样本周围及背面的PBS溶液。注意：为了降低背景，载玻片在用PBS洗一遍后，可再用含0.1% Triton X-100和5 mg/mL BSA的PBS洗3次，每次5 min，这样可将游离的未反应标记物清除干净。

8. 样本在染色缸中染色，在黑暗中将载玻片浸入装有PI溶液（1 μg/mL，用PBS新鲜配制并稀释）的染色缸，室温放置5 min。可选操作：样本在染色缸中染色，在黑暗中将载玻片浸入装有DAPI溶液（2 μg/mL，用PBS新鲜配制并稀释）的染色缸，室温放置5 min。

9. 洗涤样本，将载玻片浸入去离子水中，室温放置5 min，重复2次，总共洗3次。

10. 叩干载玻片上多余的水并且用吸水纸擦拭细胞周边的区域。

11. 立即在荧光显微镜下分析样本，用标准的荧光过滤装置在520±20 nm的荧光下观察绿色荧光；在＞620 nm下观察PI的红色荧光，或在460 nm观察蓝色的DAPI。如有必要，载玻片能在4℃黑暗条件下存放过夜。PI/DAPI能将凋亡和未凋亡的细胞都染成红色/蓝色，只在凋亡的细胞核中才有FITC-12-dUTP掺入而定位的绿色荧光。

四、利用流式细胞术检测悬浮细胞

1. 将3-5×10⁶个细胞用PBS在4℃离心（300×g）洗两次，然后重悬在0.5 mL PBS中。

2. 固定细胞，加入5 mL 1%配制于PBS中的多聚甲醛溶液，冰上放置20 min。

3. 细胞在4℃，300×g离心10 min，去上清并且重悬于5mL PBS。重复洗一次，并用0.5 mL PBS重悬细胞。

4. 通透细胞，加入5 mL冰上预冷的70%乙醇，在-20℃孵育4小时。细胞能在70%乙醇中-20℃条件下保存一周，或者，细胞可用配制于PBS中的0.2% Triton X-100溶液通透，室温放置5 min。

5. 细胞在300×g离心10 min，并用5 mL PBS重悬。重复离心，并1 mL PBS重悬。

6. 转移2×10⁶个细胞至一个1.5 mL的微量离心管。

7. 300×g离心10 min，去上清，并用80 μL 1×Equilibration Buffer重悬。室温孵育5 min。

8. 在平衡细胞的同时，在冰上融解FITC-12-dUTP标记混合物，并且依照表1，准备足够量的用于所有反应的TdT孵育缓冲液。对于2×10⁶个细胞的一个标准反应，其体积是50 μL，用50μl乘上反应数目来确定所需TdT孵育缓冲液的总体积。

9. 细胞在300×g离心10 min，去上清并把沉淀重悬在50 μL TdT孵育缓冲液中，37℃孵育60 min，避光。每隔15 min用微量移液器轻轻重悬细胞。

10. 加入1mL ,20 mM EDTA终止反应，用微量移液器轻柔混匀。

11. 300×g离心10 min，去上清并把沉淀重悬在1mL配制于PBS中的0.1% Triton X-100溶液，其中含5 mg/mL BSA，重复一次，总共洗2次。

12. 300×g离心10 min，去上清并把细胞沉淀重悬在0.5 mL PI溶液（1 μg/mL）中，其中包含250 μg 无DNA酶的Rnase A。

13. 在黑暗中室温孵育细胞30 min。

14. 用流式细胞仪分析细胞，测量520±20 nm的FITC-12-dUTP的绿色荧光和＞620 nm的PI红色荧光。PI将凋亡和未凋亡的细胞都染成红色，只在凋亡细胞核中才有FITC-12-dUTP掺入而定位的绿色荧光。

相关产品

   HB210715

Q：共染之后TUNEL 的信号就不在核内了，感觉都弥散了。TUNEL 染色就按照说明书来的，DAPI 之前孵育另外的一抗？

A：可能是后期洗涤次数过多，建议减少洗涤次数，或者洗涤动作轻柔一些。

Q：TUNEL 可以和 DAPI 一起染色细胞吗?

A：可以。

Q：Annexin V 和 JC-1、Tunel 细胞凋亡检测的区别？

A： Annexin V 是检测细胞早期凋亡的试剂，JC-1 是检测细胞中期凋亡的试剂、Tunel 是检测细胞晚期凋亡的试剂。

Q：Annexin V 和JC-1、Tunel 细胞凋亡检测的可以应用到植物或是细菌（原核生物） 吗？

A：可以，但是需要制备原生质体，因为植物细胞或是细菌（原核生物）含有细胞壁，具体的染液使用剂量只需浸没细胞即可，染色时间对于不同细胞有一定的不同。

Q：Tunel 细胞凋亡检测，细胞爬片好凋亡处理后需要在固定通透吗？

A：需要通透，因为 TdT 酶需要经过通透的细胞才能进入细胞内，而 Annexin V 和 JC-1 是不能进行染色固定的。

Q：Tunel 细胞凋亡检测时，贴壁细胞必须要先消化下来再染色吗？

A：不需要，对于贴壁细胞，要先用PBS 洗 2-3 次，然后直接用多聚甲醛固定细胞， 通透处理，染色观察。

Q：固定时间可以增加吗？

A： 4℃放置25 min左右，选择4%多聚甲醛作固定液，乙醇、甲醇、酸性固定液，会导致标记效率低；固定时间不宜过长，过长导致交联程度过高，进而降低标记效率。

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