Overview
| Product name | Annexin V-Alexa Fluor488/PI 凋亡检测试剂盒 |
| Catalog no | FXP022-020 |
| Size | 20 Tests |
Applications
| 储存条件 | 2-8℃避光保存，勿冰冻。 | ||||||||||||||||||||||||
| 有 效 期 | 一年。 | ||||||||||||||||||||||||
| 注意事项 | 本试剂盒需使用流式细胞仪进行检测。 此产品仅供研究，不用于临床诊断。 | ||||||||||||||||||||||||
| 操作步骤 | 使用本试剂盒时，需自备1×PBS缓冲液。 1. 细胞样品的准备： 对于贴壁细胞： 1. 取细胞培养瓶，用移液器小心地吸取细胞培养液，转移到一个干净的离心管内备用； 2. 用不含EDTA的胰蛋白酶消化细胞，至细胞可以被移液器轻轻吹打下来时，加入收集的细胞培养液，终止消化； 3. 用移液器吹打下所有的贴壁细胞，并轻轻吹散细胞； 4. 将细胞收集到离心管内，1000rpm左右离心5min，沉淀细胞(根据待测细胞特性，可适当调整离心时间或离心力)； 5. 用移液器小心吸除上清，可保留约50μl左右的培养液，避免吸走细胞； 6. 加入约1ml 4℃预冷的1×PBS缓冲液重悬细胞，再次离心沉淀细胞，小心吸除上清； 对于悬浮细胞： 1. 将细胞收集到离心管内，1000rpm左右离心5min，沉淀细胞(根据待测细胞特性，可适当调整离心时间或离心力)； 2. 用移液器小心吸除上清，可保留约50μl左右的培养液，避免吸走细胞； 3. 加入约1ml 4℃预冷的1×PBS缓冲液重悬细胞，再次离心沉淀细胞，小心吸除上清； 4. 根据实验用量，按1:3的比例用去离子水将4×结合缓冲液稀释成1×结合缓冲液，用于后续实验； (e.g. 若需要16ml 1×结合缓冲液，则取4ml 4×结合缓冲液+12ml去离子水)； 5. 用1×结合缓冲液重悬细胞，将细胞密度调节至1-5×106/ml； 6. 取100μl的细胞悬液于5ml流式管中，加入5μl rh Annexin V Alexa Fluor 488，用移液器轻柔吹吸混匀，室温避光孵育5min； 7. 加入10μl PI，并加400μl 1×PBS缓冲液，立刻进行流式检测。 注：实验过程中应注意轻柔吹吸细胞，避免造成细胞机械损伤；加入步骤5中的PI溶液和1×PBS缓冲液后，建议立即上机检测，若无法立即上机，应将细胞置于4℃条件下避光保存，并在1h内上机检测，避免长时间放置导致检测结果不准确。 | ||||||||||||||||||||||||
| 实验设计 | 注：为保证仪器参数及荧光补偿的正确调节，空白管和单染管中的待测细胞应同时包括凋亡细胞和正常细胞。若无法确认待测细胞的凋亡程度，可通过热刺激诱导或凋亡诱导剂诱导凋亡后再用于空白管和单染管的检测。 | ||||||||||||||||||||||||
| 样本分析 | Alexa Fluor 488的最大激发波长是488nm，最大发射波长是519nm，建议选择FL1通道检测；PI-DNA复合物的最大激发波长是535nm，最大发射波长为615nm，PI的红色荧光在FL2或FL3通道检测。用FlowJo等软件进行分析，绘制双色散点图（two-color dot plot），Alexa Fluor488为横坐标，PI为纵坐标。典型的实验中，细胞可以分成三个亚群，活细胞仅有很低强度的背景荧光，早期凋亡细胞仅有较强的绿色荧光，晚期凋亡或坏死的细胞有绿色和红色荧光双重染色。 Jurkat细胞用紫外诱导凋亡后用Annexin V Alexa Fluor 488/PI双染流式分析图谱 | ||||||||||||||||||||||||
| 常见问题 | 1. Annexin V/ PI凋亡检测的试剂盒能否检测人以外其他动物的细胞凋亡情况？ 可以。因为Annexin V是与磷脂酰丝氨酸(PS)亲和，而PS在不同种属间没差异。在正常细胞中，PS只分布在细胞膜脂质双层的内侧，而在细胞凋亡早期，PS由脂膜内侧翻向外侧。 1. 贴壁细胞做凋亡用胰酶消化下来对细胞膜损伤？ 低浓度胰酶消化，轻柔吹打贴壁细胞2～3次，离心机4℃ 1000rpm离心5min，处理得当的话，胰酶造成损伤可以控制在5%以内，有对照组的情况下对实验结果不会造成明显影响。 1. 贴壁细胞可以先染PI然后再消化下来吗？这样是否可以减小由于消化液造成的细胞膜破损而染上的PI的误差？ 先染PI可能会导致两个问题：1、不易判断每组PI是否染色均匀，2、PI对细胞有毒性，对实验结果影响比胰酶消化更大。因此不建议先染PI。 1. 为什么只能用不含EDTA的胰酶消化贴壁细胞，用含EDTA的胰酶消化细胞对结果有什么影响？ 因为Annexin V是Ca依赖的蛋白，若加入EDTA，ETDA会螯合Ca2+离子，从而影响Annexin V与PS的结合，进而影响结果。 1. 有些厂家说明书Annexin V和PI一起加？为什么你们先加Annexin V后加PI？ 用流式检测凋亡时，PI受时间的影响很大，因标记了PI后会加大细胞毒性，随着时间延长会导致PI的染色增加，特别是检测早期凋亡时，如果时间延长除了会导致在流式细胞仪上的细胞分群差距加大外，误差会明显加大。一般PI加上后立刻上机，在一个小时内检测完成。两种方法都可以，但是按照我们操作步骤造成的误差会更小。 1. 你们哪种凋亡检测试剂盒能用于转染GFP细胞的凋亡检测？ FXP147 Annexin V- Alexa Fluor 647/7AAD 凋亡检测试剂盒 FXP023 Annexin V-Alexa Fluor 647/PI 凋亡检测试剂盒 FXP027 Annexin V- PE/7AAD 凋亡检测试剂盒 | ||||||||||||||||||||||||
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image of Annexin V-Alexa Fluor488/PI 凋亡检测试剂盒
Annexin V-Alexa Fluor 488/PI 凋亡检测试剂盒
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¥580.00
Annexin V-Alexa Fluor488/PI 凋亡检测试剂盒
规格：
货期：现货
