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Annexin V-FITC/7-AAD Apoptosis Detection Kit
12个月
北京义翘神州科技股份有限公司
2℃ - 8℃
20 Tests/100 Tests
规格: | 20 Tests | 产品价格: | ¥500.0 |
---|---|---|---|
规格: | 100 Tests | 产品价格: | ¥1800.0 |
产品信息:
应用:流式
储存及稳定性:2℃-8℃避光保存,勿冷冻。
本试剂盒所有组份依保存条件储存,自收到之日起一年有效。
安全注意事项:7-AAD是一种潜在的致癌物。为了您的安全和健康,操作时请穿实验服、戴手套、做好眼睛及面部防
护,避免7-AAD接触皮肤及眼睛。
试剂盒组分:
名称 | 规格(20 Tests) | 规格(100 Tests) | 每次使用量 | 缓冲液成分 |
Annexin V-FITC | 0.1 mL | 0.5 mL | 5 ul | 含0.5% BSA、0.03% Proclin300的水 溶液 |
7-AAD | 0.1 mL | 0.5 mL | 5 ul | 含0.5% BSA、0.03% Proclin300的水 溶液 |
10×Binding Buffer | 10 mL | 50 mL | 请参阅染色 | Aqueous buffered solution containing no preservative |
应用验证:
Annexin V-FITC/7-AAD 凋亡检测试剂盒在喜树碱处理过的Jurkat细胞上做常规验证。Annexin V与磷脂酰丝氨酸(PS)的结合为钙离
子依赖性的,在反应时需使用含钙离子的缓冲液。
Annexin V-FITC/7-AAD 凋亡检测试剂盒未在贴壁细胞上做常规验证。在贴壁细胞的收集过程中,消化细胞容易造成细胞膜损伤,从
而导致产生错误的实验结果。在您的研究中如果用到贴壁细胞,建议提前摸索细胞消化条件,并且尽量避免使用含EDTA的细胞消化
液,以免产生错误的实验结果。
流式细胞术检测Annexin V-FITC染色。Annexin V-FITC及7-AAD在未处理的Jurkat细胞(上图)、4μM喜树碱(Sigma,货号C9911)处
理5小时(中图)及18小时(下图)的Jurkat细胞上的染色结果。未处理的细胞主要为Annexin V-FITC及7-AAD阴性。喜树碱处理5小时后,
主要出现两群细胞:未凋亡的活细胞( Annexin V-FITC 、7-AAD双阴性),早期凋亡的细胞( Annexin V-FITC 阳性、7-AAD阴性)。
一小部分Annexin V-FITC、7-AAD双阳性的细胞为凋亡晚期及已经死亡的细胞。经过18小时的处理后,凋亡晚期及已经死亡的细胞
( Annexin V-FITC、7-AAD双阳性)比例明显增加。
染色步骤
微量液体建议开盖前瞬时离心
1. 收集细胞,用4℃预冷的PBS洗细胞2次,离心去上清后用1×Binding Buffer重悬细胞至细胞密度为0.1×107~1×107 个/mL。
1×Binding Buffer : 1份10×Binding Buffer中加入9份蒸馏水,混匀,现用现配。
2. 将细胞悬液转移至流式管中,每管100 μL.
3. 每管细胞中加入5 μL Annexin V-FITC及5 μL 7-AAD,轻轻混匀,室温(25℃)避光孵育15min。
4. 加入400 μL 1X Binding Buffer终止反应,1小时内上机检测。
注意事项
1. 为了得到准确的实验结果,建议设置以下对照:
a) 阴性对照:未染色的细胞。
b) 单染对照:只染Annexin V-FITC(无7-AAD)的细胞,只染7-AAD(无Annexin V-FITC)的细胞。
阴性对照和单染对照用于设置流式细胞仪电压及补偿。
c) 实验对照:未处理的细胞,用于确定本底水平的凋亡及死亡的细胞比例。
d) 选做对照:为确定Annexin V-FITC的结合特异性,在加入Annexin V-FITC之前,可用纯化的Annexin V(10448-HNAE)封闭
Annexin V-FITC的结合位点。
2. 不要使用保质期外的试剂。
3. 避免混合使用不同批次的试剂。
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