使用方法

1）细胞样品制备：细胞数量控制在1×10⁵~1 × 10⁶个。

a）贴壁细胞：小心吸除细胞培养液，用胰酶消化细胞，制备成单细胞悬液。1000 g离心5 min，沉淀细胞，弃上清，用1 mL预冷的PBS润洗细胞一次，离心收集细胞。

b）悬浮细胞：1000 g离心5 min，沉淀细胞，小心吸除上清。加入1 mL预冷的PBS，重悬细胞，再次离心收集细胞。

c）组织细胞：将组织块用剪刀剪成尽量小的小块后，用0.25%的胰酶消化0.5-1 h，经过200-400目筛网过滤得到单细胞悬液。1000 g离心5 min，沉淀细胞。加入约1 mL预冷的PBS，重悬细胞，再次离心沉淀细胞。如组织难以消化，可加入适量胶原酶。

2）细胞固定：细胞沉淀用1 mL预冷的70%乙醇轻轻混匀，4℃固定2 h以上或者过夜。接下来1000 g，离心5 min沉淀细

胞后，用1 mL预冷的PBS重悬。然后再次1000 g离心5 min沉淀细胞。

3）染色：

在0.5 mL染色缓冲液(40301-C)中加入10 μL 碘化丙啶储液（40301-B）和10 μLRNase A（40301-A）溶液，混匀待用。每个细胞样品加入0.5 mL配置好的碘化丙啶染色液，轻轻混匀重悬细胞。37℃避光孵育30 min，就可以进行流式检测，流式检测最好在5 h内完成。

【注】：配置好的PI染色液在短时间内可以4℃保存，宜当日使用。

4.流式检测和分析：细胞用400目筛网过滤，用流式细胞仪进行检测，在激发波长488 nm波长处检测，同时检测光散射情况。采用适当分析软件进行细胞DNA含量分析和光散射分析。

HB221122

Q：这款试剂盒是不是凋亡和周期均可以检测？

A：这款试剂盒是检测细胞周期的，细胞周期检测中细胞内的DNA被PI染色后，可以用流式细胞仪对细胞进行DNA含量测定，根据DNA含量的分布情况，进行细胞周期和细胞凋亡分析。

Q：Staining Solution能否使用PBS或DPBS代替？

A：可以

Q：流式周期结果如何分析？G1,S，G2-M数据分析。G2/G1的数据是有意义吗？

A：一般是G1期数据都基本没有变化，抑制细胞周期的药物处理就是G2/M期增加，S期减少。一般是看各个时期的比值

Q：流式周期固定的时候能否直接加70-75%的乙醇重悬细胞固定？

A：不可以，如果直接用70-75%的乙醇，会导致固定效果差或者出现细胞固定后无沉淀的现象。建议先用PBS重悬细胞后，再加入无水乙醇稀释到70-75%。

Q：做流式周期的时候，只出现单峰是什么原因？是不是你们的试剂有问题？

A：试剂是没有问题的，能出现峰说明试剂是可以和核酸结合的，只出现单峰或没有G2期，可能原因有以下几点：1.细胞状态，是不是细胞状态较差，建议调整细胞状态之后进行实验。2.细胞密度过密，如果细胞接种的时候过密会导致细胞接触抑制。3.原代细胞，有些原代细胞是没有增值能力的。4.圈门没圈好，建议正确圈门

Q：流式分析细胞周期，收集了10的6次方细胞，但细胞在乙醇固定之后，还可以看到细胞沉淀的，但PBS洗涤两次之后，就基本没什么细胞沉淀了，上机发现看不到细胞了。这是怎么回事啊？怎么解决这个问题啊？

A：1. 先用冷PBS悬浮细胞，充分悬浮，使细胞充分分散成单细胞。之后缓慢加入预冷的无水乙醇，终浓度为70-75%乙醇。不直接加75%乙醇的原因是：直接加入乙醇会导致细胞团聚的现象，很难重悬成单细胞。乙醇固定之后没有细胞沉淀。

2.很可能是洗细胞的过程中丢失了，解决办法有：采用尖底的离心管和水平离心机。离心后尽量用吸管吸取上清，不要倾倒；吸上清时残留1mm左右的水膜，不要吸完。离心的转速或时间可稍微增加一点儿。每次加溶液时，吸头最好不要接触液面；混匀时最好不要用吸头吹打，以免吸头挂壁带走部分细胞。

Q：试剂盒的 RNase A和 PI的浓度是多少，Staining Solution成分是什么？

A:RNase A和 PI的浓度都是1mg/ml，Staining Solution成分是一些盐溶液配方，配方不告知，用完了可以暂用PBS替代。

Q：周期的各个时期加起来不足100%是什么原因呢？

A：应该是没调好，因为流式是一个动态的过程，首位有点偏差是正常的，差的大可能是圈了一些黏连的细胞，需要去掉这些细胞，或者选择不同的拟合公式，可以调整圈门看看。

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产品描述

细胞周期与细胞凋亡检测试剂盒（Cell Cycle and Apoptosis Analysis Kit）采用了经典的碘化丙啶染色(Propidium staining，即PI staining)方法对细胞周期与细胞凋亡进行分析。

碘化丙啶 (Propidium，PI) 是一种双链DNA荧光染料，其嵌入双链DNA后可以产生荧光，并且荧光强度和双链DNA的含量成正比。细胞内的DNA被PI染色后，可以用流式细胞仪对细胞进行DNA含量测定，根据DNA含量的分布情况，进行细胞周期和细胞凋亡分析。

PI染色后，假设G0/G1期细胞的荧光强度为1，那么含有双份基因组DNA的G2/M期细胞的荧光强度的理论值为2，正在进行DNA复制的S期细胞的荧光强度为1-2之间。凋亡细胞由于细胞核发生浓缩以及发生DNA片段化 (DNA fragmentation) 导致部分基因组DNA片断在染色过程中丢失，因此凋亡细胞PI染色后呈现明显的弱染，即荧光强度小于1，在流式检测的荧光图上出现所谓的sub-G1峰，即凋亡细胞峰。

细胞凋亡也可以用流式细胞仪观察细胞光散射的变化来检测。 细胞发生凋亡时，由于胞浆和染色质浓缩、核碎裂，产生凋亡小体，使细胞的光散射性质发生变化。凋亡前期，染色质皱缩，细胞密度增加，前向角光散射色显著降低。凋亡后期，细胞产生凋亡小体，前向角光散射和侧向角光散色都显著降低。

本试剂盒通常应用于贴壁或悬浮细胞的细胞周期与细胞凋亡检测。如果用于组织的细胞周期与细胞凋亡检测，则必须把组织消化成单细胞状态，才可以进行检测。

产品组分

运输与保存方法

运输：冰袋（wet ice）运输。

保存方法：-20℃避光保存，有效期为2年。

【注】如果需要在短时间内多次重复使用，可以于4℃避光保存，2个月内有效。

注意事项

1）本试剂盒需要使用流式细胞仪进行检测。

2）细胞处理需轻柔，尽量避免人为的损伤细胞。

3）为防止不同批次细胞在实验时所处周期不同导致重复性差，可以在实验前进行细胞的同步化处理。实验细胞应处于对数

生长期，贴壁细胞一般在50～80%汇合度时收集为宜。

4）400目筛网过滤是用来将粘在一起的细胞团滤掉，留下单细胞，否则会出现人为的多倍体干扰。如果没有条件过滤，请在染色之前将细胞轻弹以分散，再进行染色。

5）荧光染料均存在淬灭问题，保存和使用过程中请尽量注意避光，以减缓荧光淬灭。

6）操作碘化丙啶时，应注意防护，保护眼睛、避免吸入。

7）为了您的安全和健康，请穿实验服并戴一次性手套操作。

8) 本产品仅作科研用途！

 产品描述

依托泊甙，是一种抗肿瘤试剂，作为一种凋亡诱导剂，依托泊甙是一种拓扑异构酶II抑制剂（IC₅₀=59.2 μM），常用工作浓度5-50 µM。

产品性质

运输和保存方法

冰袋运输。-20℃保存，冻干粉形式的Etoposide可以稳定保存24个月；液体形式的Etoposide建议在6个月内使用完，分装避免反复冻融。 

注意事项

1）为了您的安全和健康，请穿实验服并戴一次性手套操作。

2）本产品仅作科研用途！

HB221011

Q：可以用来哪些肿瘤细胞凋亡呢？

A：小细胞肺癌，恶性淋巴瘤，恶性生殖细胞瘤，白血病，对神经母细胞瘤，横纹肌肉瘤，卵巢癌，非小细胞肺癌，胃癌和食管癌等

[1] Li Y, Sun H, Zhang C, et al. Identification of translationally controlled tumor protein in promotion of DNA homologous recombination repair in cancer cells by affinity proteomics. Oncogene. 2017;36(50):6839-6849. doi:10.1038/onc.2017.289(IF:7.519)