Annexin v-apc conjugate

Annexin V（膜联蛋白-V）是一种分子量为35-36 KD的Ca2+依赖性磷脂结合蛋白，可与磷脂酰丝氨酸（PS）选择性结合。磷脂酰丝氨酸（PS）主要分布在细胞膜内侧，即与细胞浆相邻的一侧。在细胞发生凋亡的早期，不同类型的细胞都会把磷脂酰丝氨酸外翻到细胞表面，暴露在细胞外环境中。此时，使用荧光蛋白APC标记的 Annexin V，即 Annexin V-APC，与外翻的磷脂酰丝氨酸（PS）结合，就可用流式细胞仪直接检测到磷脂酰丝氨酸的外翻这一细胞凋亡的重要特征。正常细胞不会被Annexin V-APC所染色，发生凋亡或坏死的细胞会被Annexin V-APC所染色。Annexin V-APC可以与部分非渗透性的细胞核染料 ( 7-AAD/PI ) 等联合使用，区分处于不同凋亡时期的细胞。 产品参数： Annexin V-APC：Ex/Em = 650/660 nm 注意事项： 1. 使用前请将产品瞬时离心至管底，再进行后续实验。 2. 为降低细胞凋亡进程，孵育过程可在冰上操作，但孵育时间至少延长至30 min。 3. 由于细胞凋亡是一个快速的过程，建议样品在染色后 1 h之内进行分析。 4. 对于贴壁细胞，消化是一个关键步骤。贴壁细胞诱导细胞凋亡时如有漂浮细胞，需收集漂浮细胞和贴壁细胞后合并染色。处理贴壁细胞时要小心操作，尽量避免人为的损伤细胞。胰酶消化时间过短，细胞需要用力吹打才能脱落，容易造成细胞膜的损伤，PI 摄入过多；消化时间过长，细胞膜同样易造成损伤，甚至会影响细胞膜上磷脂酰丝氨酸与 Annexin V-APC的结合。消化时将胰酶铺满孔板底后，轻摇时胰酶与细胞充分接触，然后倒掉大部分胰酶，利用剩余的少量胰酶再消化一段时间，待细胞间空隙增大，瓶底呈花斑状即可终止。在消化液中尽量不用 EDTA，EDTA 会影响 Annexin V 与 PS 的结合。 5. 贴壁细胞用胰蛋白酶消化后，建议在最佳培养条件和培养基中恢复约30 min后染色，避免假阳性。 6. 为了避免洗涤细胞时损失细胞，在吸液时可以用大的 Tip 头套上小的 Tip 头吸液。 7. 染料的最佳使用浓度由具体实验要求确定。 8. 荧光染料均存在淬灭问题，保存和使用过程中请尽量注意避光，以减缓荧光淬灭。 9. 为了您的安全和健康，请穿实验服并戴一次性手套操作。 应用范围： 早期凋亡检测，Annexin V 偶联荧光蛋白 Product introduction： Annexin V (annexin-V) is a ca2+ - dependent phospholipid binding protein with a molecular weight of 35-36 KD, which can selectively bind to phosphatidylserine (PS). Phosphatidylserine (PS) is mainly distributed on the inner side of the cell membrane, that is, the side adjacent to the cytoplasm. At the early stage of apoptosis, different types of cells will evert phosphatidylserine to the cell surface and expose it to the extracellular environment. At this time, annexin V labeled with fluorescent protein APC, namely annexin v-apc, is combined with everted phosphatidylserine (PS), and the everted phosphatidylserine, an important feature of apoptosis, can be directly detected by flow cytometry. Normal cells will not be stained by annexin v-apc, and apoptotic or necrotic cells will be stained by annexin v-apc. Annexin v-apc can be combined with some non permeable nuclear dyes (7-aad/pi) to distinguish cells at different stages of apoptosis. Product parameters： Annexin v-apc:ex/em = 650/660 NM Matters needing attention： 1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. to reduce the process of apoptosis, the incubation process can be operated on ice, but the incubation time should be extended to at least 30 min. 3. as apoptosis is a rapid process, it is recommended that samples be analyzed within 1 h after staining. 4. for adherent cells, digestion is a key step. If there are floating cells when adherent cells induce apoptosis, the floating cells and adherent cells should be collected and stained. Handle adherent cells with care to avoid artificial damage to cells. The trypsin digestion time is too short, and the cells need to be blown hard to fall off, which is easy to cause damage to the cell membrane and excessive PI intake; If the digestion time is too long, the cell membrane is also prone to damage, and even affect the binding of phosphatidylserine and annexin v-apc on the cell membrane. When digesting, spread pancreatin on the bottom of the well plate, fully contact the pancreatin with the cells when shaking gently, then pour out most of the pancreatin, use the remaining small amount of pancreatin to digest for a period of time, and terminate when the gap between cells increases and the bottom of the bottle is spotted. Try not to use EDTA in the digestive juice, which will affect the binding of annexin V to PS. 5. after the adherent cells are digested with trypsin, it is recommended to stain after recovering in the optimal culture conditions and medium for about 30 min to avoid false positives. 6. in order to avoid losing cells when washing cells, you can use a large tip over a small tip to aspirate. 7. the optimal concentration of dye is determined by the specific experimental requirements. 8. fluorescent dyes have quenching problems. Please try to avoid light during storage and use to slow down fluorescence quenching. 9. for your safety and health, please wear experimental clothes and disposable gloves. Scope of application： Early apoptosis detection, annexin v-coupled fluorescent protein

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