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。
注意事项：
1. 1. 使用时请避光以减缓荧光淬灭；
2. 7-AAD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。
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 cells /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2 μL Annexin V-AF488及5 μL 7-AAD染料；
4. 加入100 μL结合缓冲液， 轻柔涡旋混匀细胞，室温避光孵育10 min；
5. 1h内完μL成上机分析。
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.
Precautions
1. Please try to avoid light when using to slow down the quenching of fluorescence.
2. 7-AAD Staining 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 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 0.5-1.0x10⁶ cells /mL .
3. Add 2 µL Annexin V-AF488 and 5 µL 7-AAD to 100 µL of the cell suspension.
4. Add 100 µLof 1x Binding Buffer to each assay. Gently vortex the cells and incubate for 10 min at RT (25°C) in the dark.
5. Analyze by flow cytometry within 1 hr.
ZJ25F1230226
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