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。
膜联蛋白（Annexins）是一类钙离子依赖型磷脂结合蛋白，能特异性结合磷脂酰丝氨酸（PS）。在正常生理状态下，PS只分布在细胞膜脂质双层的内侧。细胞凋亡早期， PS由脂膜内侧翻向外侧，在磷脂双分子层中的不对称分布丧失，从而使细胞成为被吞噬的目标。一旦PS暴露在膜的外表面，它便可在钙离子存在的情况下，被荧光标记的膜联蛋白V（Annexin V）检测到。
在凋亡早期，活性染料（如碘化丙啶PI、7-AAD）不能通过细胞脂膜。这类细胞便只会被Annexin V染色，但不被活性染料染色，从而可区分出处于早期凋亡的细胞。然而，在凋亡晚期，细胞膜完整性丧失，使得Annexin V也能结合细胞内部的PS。此时，活性染料可用于区分晚期凋亡和坏死细胞（Annexin V阳性、活性染料阳性）与早期凋亡细胞（Annexin V阳性、活性染料阴性）。
本试剂盒适用于通过流式细胞术对死亡细胞（如凋亡或坏死细胞）进行鉴定和计数。
A1372286 | 组份 | 20T | 50T | 100T | 储存条件 | 单次测试使用量 |
A1372286A | 10x Annexin V 结合缓冲液 | 5 mL | 10 mL | 20 mL | 2-8℃ | 200 μL/0.5-1.0x10⁵细胞 |
A1372286B | Annexin V-FITC | 100 μL | 250 μL | 500 μL | 2-8℃, 避光. | 5 μL/0.5-1.0x10⁵细胞 |
A1372286C | PI染色液 | 100 μL | 250 μL | 500 μL | 2-8℃, 避光. | 2 μL/0.5-1.0x10⁵细胞 |
注: 单次测试推荐染色的细胞数量为 0.5-1.0x10⁵细胞
使用说明：
1. 使用前需将10x结合缓冲液用去离子水稀释至1x (1 mL 10x缓冲液加9 mL去离子水)；
2. 预冷PBS清洗细胞两次后，按照0.5-1.0x106/mL的密度用1x Annexin V结合缓冲液重悬；
3. 每100 μL细胞悬液中加入5 μL Annexin V-FITC及2μL PI染料；
4. 轻柔涡旋混匀细胞，室温避光孵育10min；
5. 加入100 μL结合缓冲液，1h内完成上机分析。
注意事项：
1. 使用时请避光以减缓荧光淬灭；
2. PI染料具有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。
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).
This kit is suitable for the identification and enumeration of dead cells, such as apoptotic or necrotic cells, by flow cytometry.
A1372286 | Components | 20T | 50T | 100T | Storage | Quantity Per Test |
A1372286A | 10x Annexin V Binding Buffer | 5mL | 10mL | 20mL | 2-8℃ | 200 μL/0.5-1.0x10⁵ cells |
A1372286B | Annexin V-FITC | 100 μL | 250 μL | 500 μL | 2-8℃. Store in the dark. | 5 μL/0.5-1.0x10⁵ cells |
A1372286C | PI Staining Solution | 100 μL | 250 μL | 500 μL | 2-8℃. Store in the dark. | 2 μL/0.5-1.0x10⁵ cells |
Note: The recommended number of cells to stain per test is 0.5-1.0x10⁵ cells.
Instruction for use
1. Dilute 10x Binding Buffer 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 amount of cells in Annexin V Binding Buffer at a concentration of 0.5-1.0 x 106 cells/mL.
3. Add 5 µl of Annexin-FITC and 2 µl PI to 100 µL of the cell suspension
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.
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.
A1372286 | 组份 | 20T | 50T | 100T | 储存条件 | 单次测试使用量 |
A1372286A | 10× Annexin V 结合缓冲液 | 5 mL | 10 mL | 20 mL | 2-8℃ | 200 μL/0.5-1.0x10⁵细胞 |
A1372286B | Annexin V-FITC | 100 μL | 250 μL | 500 μL | 2-8℃, 避光 | 5 μL/0.5-1.0x10⁵细胞 |
A1372286C | PI染色液 | 100 μL | 250 μL | 500 μL | 2-8℃, 避光 | 2 μL/0.5-1.0x10⁵细胞 |
Annexin V-FITC/PI Apoptosis Detection Kit (A1372286) - Flow Cytometry
Flow cytometric analysis of U937 cells untreated (left) or treated with 4µM camptothecin (C111281) for 4 hours using Annexin V-FITC/PI Apoptosis Detection Kit (A1372286).
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F1039963 |
分析证书 | A1372286 |
| ZJ26F0737301 |
分析证书 | A1372286 |
| ZJ26F0737302 |
分析证书 | A1372286 |
| ZJ26F0737303 |
分析证书 | A1372286 |
| ZJ26F0434047 |
分析证书 | A1372286 |
| ZJ26F0434048 |
分析证书 | A1372286 |
| ZJ26F0434046 |
分析证书 | A1372286 |
| ZJ25F1230235 |
分析证书 | A1372286 |
| ZJ25F1230236 |
分析证书 | A1372286 |
| ZJ25F1230230 |
分析证书 | A1372286 |
| ZJ25F0826142 |
分析证书 | A1372286 |
| ZJ25F0826141 |
分析证书 | A1372286 |
| ZJ25F0826140 |
分析证书 | A1372286 |
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