BioReagent,生物染色剂,用于显微镜,适用于免疫荧光(IF) 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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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。
背景介绍
Annexin V 是一种钙离子依赖性磷脂结合蛋白，与磷脂酰丝氨酸 (PS) 有高度亲和力。当细胞发生凋亡时，膜内侧的磷脂酰丝氨酸 (PS) 外翻到膜表面，而被荧光染料标记的Annexin V结合，可通过流式细胞仪或荧光显微镜进行检测。Annexin V 搭配 PI 或 7-AAD、DAPI 等 DNA 染色液使用，可区分处于不同凋亡时期的细胞。我们提供了一系列膜联蛋白 V 和荧光染料的偶联物，包括AF405、AF488、AF647、AF680、AF750和膜联蛋白 V 偶联物以及 Annexin V APC、生物素、FITC、Pacific Blue、 PE 、PE-Cy5、PE-Cy7偶联物。这些偶联物可产生较亮信号，适用于所有可用激光，可用作独立试剂或与细胞核染色液组合成试剂盒使用。
注意事项：
1. 使用时请避光以减缓荧光淬灭；
2. PI染料具有剧毒性和致突变性，操作时需谨慎；
3. 由于膜联蛋白V（Annexin V）与磷脂酰丝氨酸（PS）的结合具有钙依赖性，因此在Annexin V实验中务必避免使用含EDTA或其他钙螯合剂的缓冲液。
使用说明：
1. 使用前需将10x 结合缓冲液（A1372288）用去离子水稀释至1x（1mL 10x缓冲液加9mL去离子水）。
2. 预冷PBS清洗细胞两次后，按照1.0-5.0x105/mL的密度用1x Annexin V结合缓冲液重悬。
3. 每100μL细胞悬液中加入5μL 荧光标记Annexin V和细胞核活性染料，例如PI (P1373641; P1372285), 7-AAD (A1372406), 或者DAPI (D1372407)等。
4. 轻柔涡旋混匀细胞，室温避光孵育10min。
5. 加入100μL结合缓冲液，1h内完成上机分析。
Background information
Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet marking cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by fluorescently labeled Annexin V in a calcium-dependent manner.
In early-stage apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI), 7-AAD. These cells will stain with Annexin V but not a viability dye, thus distinguishing cells in early apoptosis. However, in late stage apoptosis, the cell membrane loses integrity thereby allowing Annexin V to also access PS in the interior of the cell. A viability dye can be used to resolve these late-stage apoptotic and necrotic cells (Annexin V, viability dye-positive) from the early-stage apoptotic cells (Annexin V positive, viability dye-negative).
We offer recombinant Annexin V conjugated to a numerous fluorophores, as well as an Annexin V biotin conjugate which can be detected with fluorophore-labeled streptavidin. By binding to PS, fluorophores labeled Annexin V can be used to detect and quantify apoptotic cells via flow cytometry or fluorescence microscopy. The excitation and emission maxima of the Annexin V conjugates are summarized in the following table.
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Precautions
1. Please try to avoid light when using to slow down the quenching of fluorescence.
2. Propidium Iodide Solution is toxigenic and mutagenic; handle with care.
3. Due to the calcium dependence of the Annexin V:PS interaction, it is critical to avoid buffers containing EDTA or other calcium chelators during Annexin V experiments.
Instruction for use
1. Dilute 10X Binding Buffer (A1372288) to 1X using distilled water (1 mL 10X Binding Buffer + 9 mL ddH2O).
2. Wash cells twice with cold PBS and then resuspend the desired amounts of cells in Annexin V Binding Buffer at a concentration of 1.0-5.0 x 106 cells/mL.
3. Add 5 µL of Annexin V-AF647 to 100 µL of the cell suspension. Stain with a viability dye, such as PI (P1373641; P1372285), 7-AAD (A1372406), or DAPI (D1372407) dyes, if desired.
4. Gently vortex the cells and incubate for 10 min at RT (25°C) in the dark.
5. Add 100 µL of 1X Binding Buffer to each assay. Analyze by flow cytometry within 1 hr.
Annexin V-AF647 (rp226060) - Flow Cytometry
U937 cells were either untreated (left) or treated (right) with 5µM camptothecin (C111281) for 4 hours, then stained with Annexin V-AF647 (rp226060) and propidium iodide (PI) (P1372285) in Annexin V Binding buffer (A1372288) for 10 minutes at room temperature.
5 μL/test;
For flow cytometric staining, the suggested use of this reagent is 5 µL per 100,000 - million cells in a 100 µL volume of Annexin V Binding Buffer. It
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ25F0926572 |
分析证书 | rp226060 |
| ZJ25F0926573 |
分析证书 | rp226060 |
| ZJ25F0926574 |
分析证书 | rp226060 |
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