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概述
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PE标记的 Annexin V, 即 Annexin V-PE，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-PE所染色，发生凋亡或坏死的细胞会被Annexin V-PE所染色。Annexin V-PE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。
本试剂盒中提供的RedNucleus II是一种远红光染料，属于蒽醌类化合物，不能透过活细胞和早期凋亡细胞完整的细胞膜，是非渗透性的，但是可以快速染色死亡和透化细胞中的细胞核/dsDNA。RedNucleus II是一种理想的碘化丙啶（PI）和7-AAD的替代物，与Annexin V-PE联用，无需补偿调节，具有更好的光谱特性：不受紫外线的激发，也不与PE/PE同系物重叠发射，故可以和FITC，PE及紫色荧光染料结合用于多色分析。与Annexin V-PE联用时，RedNucleus II被排除在活细胞和早期凋亡细胞外，而晚期凋亡细胞和死细胞则同时被Annexin V-PE和RedNucleus II结合染色呈现双阳性。
Annexin V-PE/RedNucleus II细胞凋亡检测试剂盒可用流式细胞仪及或其他荧光检测设备进行检测。
组分：
|
产品参数：
Annexin V-PE：Ex/Em=488/578 nm RedNucleus II：Ex/Em=635/695 nm
使用方法：
1. 实验设计：空白管：阴性对照组细胞，不加 Annexin V-PE/RedNucleus II。用于调节电压。
单染管：阳性对照组细胞，只加 Annexin V-PE/只加RedNucleus II。用于调节补偿。
检测管：处理的细胞，加 Annexin V-PE/RedNucleus II。用空白管和单染管调节好电压补偿后，获得所需要的 流式数据。
2. 收集细胞
（1）对于悬浮细胞：
a. 在进行完细胞凋亡刺激后，1000 rpm 离心 5 min，弃上清，收集细胞，用 PBS 轻轻重悬细胞并计数。
注：PBS 重悬不能省略，PBS 重悬的过程同时也起到了洗涤细胞的作用，可以保证后续 Annexin V-PE 的结合。
b. 取 5×104 - 1×105 个重悬的细胞，1000 rpm 离心 5 min，弃上清，加入 100 µL 1 ×Annexin V 结合缓冲液轻轻重悬细胞。 c. 加入 5 µL Annexin V-PE，轻轻混匀。
d. 加入 5 µL RedNucleus II 染色液，轻轻混匀。
e. 室温 ( 20-25ºC ) 避光孵育 15 min。可以使用铝箔进行避光。孵育过程中可以重悬细胞 2-3 次以改善染色效果。
（2）对于贴壁细胞：
a. 把细胞培养液吸出至一合适离心管内，PBS 洗涤贴壁细胞一次，加入适量胰酶细胞消化液 (不含 EDTA) 消化细胞。室温 孵育至轻轻吹打可以使贴壁细胞吹打下来时，吸除胰酶细胞消化液。需避免胰酶的过度消化。
注：对于贴壁细胞，胰酶消化步骤很关键。胰酶消化时间如果过短，细胞需要用力吹打才能脱落，容易造成细胞膜的损伤， 从而导致细胞坏死的假阳性；消化时间如果过长，同样易造成细胞膜损伤而出现细胞坏死的假阳性，甚至会影响细胞膜上磷 脂酰丝氨酸与 Annexin V-PE 的结合从而干扰对于细胞凋亡的检测。
b. 加入上步中收集的细胞培养液，把细胞轻轻吹打下来，转移到离心管内，1000 rpm 离心 5 min，弃上清，收集细胞，用 PBS 轻轻重悬细胞并计数。
注：加入上步中的细胞培养液非常重要，一方面可以收集已经悬浮的发生凋亡或坏死的细胞，另一方面细胞培养液中的血清 可以有效抑制或中和残留的胰酶。残留的胰酶会消化并降解后续加入的 Annexin V-PE，导致染色失败。
c. 取 5×104-1×105 个重悬的细胞，1000 rpm 离心 5 min，弃上清，加入 100 µL 1 × Annexin V 结合缓冲液轻轻重悬细胞。 d. 加入 5 µL Annexin V-PE，轻轻混匀。
e. 加入 5 µL RedNucleus II 染色液，轻轻混匀。
f. 室温 ( 20-25ºC ) 避光孵育 15 min。可以使用铝箔进行避光。孵育过程中可以重悬细胞 2-3 次以改善染色效果。
3. 结果分析：
（1）流式细胞仪检测：
a. 孵育完成后，可直接加入 400 μL 1×Annexin V 结合缓冲液重悬细胞，立即上机检测，Annexin V-PE 由 488 nm/566 nm 激 光激发，检测荧光发射光谱在 578 nm 处（BL2（FL2）/YL1 通道），RedNucleus II 通道发射光谱约在 695 nm 处（RL1（FL4） 通道）。
b. 在双变量流式细胞仪的散点图上，左下象限显示活细胞，为（Annexin V-PE-/RedNucleus Ⅱ-）；右下象限为早期凋亡细 胞，为（Annexin V-PE+/RedNucleus Ⅱ-）；右上象限是坏死与晚期凋亡细胞，为（Annexin V-PE+/RedNucleus Ⅱ+）；左上 象限显示裸核细胞，为（Annexin V-PE-/RedNucleus Ⅱ+）。
（2）荧光显微镜检测：
a. 1000 rpm 离心 5 min，收集细胞，用 400 µL 1 ×Annexin V 结合缓冲液轻轻重悬细胞。将细胞移至 96 孔板中沉降片刻或进 行细胞涂片后，置于荧光显微镜下观察。
b. Annexin V-PE 可用 PE 适用滤光片。RedNucleus II 可用远红长通滤光片。
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，RedNucleus II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-PE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 Kit
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 in the inner side of the cell membrane, that is, the side adjacent to the cytoplasm. In the early stage of apoptosis, different types of cells will turn phosphatidylserine out to the cell surface and expose to the extracellular environment. At this time, using Annexin V labeled with fluorescent protein PE, that is, Annexin V-PE, combined with phosphatidylserine ( PS ), the eversion of phosphatidylserine, an important feature of apoptosis, can be directly detected by flow cytometry. Normal cells will not be stained by Annexin V-PE, apoptotic or necrotic cells will be stained by Annexin V-PE. Annexin V-PE can be used in combination with partially non-permeable nuclear dye ( 7-AAD / PI ) to distinguish cells at different stages of apoptosis.
RedNucleus II provided in this kit is a far-red dye that belongs to an anthraquinone compound and cannot penetrate the intact cell membrane of living cells and early apoptotic cells. It is non-permeable, but can quickly stain the nucleus / dsDNA in dead and permeable cells. RedNucleus II is an ideal substitute for propidium iodide ( PI ) and 7-AAD.Combined with Annexin V-PE, it has better spectral characteristics without compensation regulation : it is not excited by ultraviolet light and does not overlap with PE / PE homologues, so it can be combined with FITC, PE and purple fluorescent dyes for multicolor analysis. When combined with Annexin V-PE, RedNucleus II was excluded from living cells and early apoptotic cells, while late apoptotic cells and dead cells were double-positive for Annexin V-PE and RedNucleus II.
Annexin V-PE / RedNucleus II apoptosis detection kit can be detected by flow cytometry or other fluorescence detection equipment.
Components:
|
Product parameters：
Annexin v-pe:ex/em=488/578 nmrednucleus ii:ex/em=635/695 NM
Usage method:
1. Experimental design: Blank tube: Negative control group cells, without Annexin V-PE/RedNucleus II. Used to regulate voltage.
Single staining tube: Positive control group cells were treated with Annexin V-PE alone/RedNucleus II alone. Used for adjusting compensation.
Detection tube: Add Annexin V-PE/RedNucleus II to the processed cells. After adjusting the voltage compensation using blank tubes and single dye tubes, obtain the required flow data.
2. Collect cells
(1) For suspended cells:
a. After inducing cell apoptosis, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, collect the cells, gently resuspend the cells in PBS, and count them.
Note: PBS resuspension cannot be omitted. The process of PBS resuspension also serves to wash cells, ensuring the subsequent binding of Annexin V-PE.
b. Take 5 × 104-1 × 105 resuspended cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add 100 µ L of 1 × Annexin V binding buffer to gently resuspend the cells. c. Add 5 µ L Annexin V-PE and mix gently.
d. Add 5 µ L of RedNucleus II staining solution and mix gently.
e. Incubate at room temperature (20-25 º C) in the dark for 15 minutes. Aluminum foil can be used to avoid light. During the incubation process, cells can be resuspended 2-3 times to improve staining efficiency.
(2) For adherent cells:
a. Suck out the cell culture medium into a suitable centrifuge tube, wash the adherent cells with PBS once, and add an appropriate amount of trypsin cell digestion solution (without EDTA) to digest the cells. Incubate at room temperature until gently blowing can remove the trypsin cell digestion solution when the adherent cells are blown down. Overdigestion of pancreatic enzymes should be avoided.
Note: For adherent cells, the trypsin digestion step is crucial. If the trypsin digestion time is too short, cells need to be blown hard to detach, which can easily cause damage to the cell membrane and lead to false positives of cell necrosis; If the digestion time is too long, it can also cause cell membrane damage and false positives of cell necrosis, and even affect the binding of phosphatidylserine and Annexin V-PE on the cell membrane, thereby interfering with the detection of cell apoptosis.
b. Add the cell culture medium collected in the previous step, gently blow down the cells, transfer them to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, collect the cells, gently resuspend the cells in PBS and count them.
Note: Adding the cell culture medium from the previous step is very important. On the one hand, it can collect cells that have already been suspended and undergone apoptosis or necrosis. On the other hand, the serum in the cell culture medium can effectively inhibit or neutralize residual trypsin. The residual trypsin will digest and degrade the subsequently added Annexin V-PE, leading to staining failure.
c. Take 5 × 104-1 × 105 resuspended cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add 100 µ L of 1 × Annexin V binding buffer to gently resuspend the cells. d. Add 5 µ L Annexin V-PE and mix gently.
e. Add 5 µ L of RedNucleus II staining solution and mix gently.
f. Incubate at room temperature (20-25 º C) in the dark for 15 minutes. Aluminum foil can be used to avoid light. During the incubation process, cells can be resuspended 2-3 times to improve staining efficiency.
3. Result analysis:
(1) Flow cytometry detection:
a. After incubation, 400 μ L of 1 × Annexin V binding buffer can be directly added to resuspend the cells, and immediately detected on the machine. Annexin V-PE is excited by 488 nm/566 nm laser, and the fluorescence emission spectrum is detected at 578 nm (BL2 (FL2)/YL1 channel), while the RedNucleus II channel emission spectrum is approximately at 695 nm (RL1 (FL4) channel).
b. On the scatter plot of the bivariate flow cytometer, live cells are shown in the lower left quadrant, which is (Annexin V-PE -/RedNucleus II -); The lower right quadrant represents early apoptotic cells, which are (Annexin V-PE+/RedNucleus II -); The upper right quadrant represents necrotic and late stage apoptotic cells, which are (Annexin V-PE+/RedNucleus II+); The upper left quadrant displays naked nuclear cells, which are (Annexin V-PE -/RedNucleus II+).
(2) Fluorescence microscopy detection:
a. Centrifuge at 1000 rpm for 5 minutes, collect cells, and gently resuspend them in 400 µ L of 1 × Annexin V binding buffer. Transfer the cells to a 96 well plate and settle for a moment or perform cell smear, then observe under a fluorescence microscope.
b. Annexin V-PE is compatible with PE filters. RedNucleus II can use a far red long pass filter.
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 intake of rednucleus II; 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-pe 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 Kit
规格
英文名称
Annexin v-pe / rednucleus Ⅱ apoptosis Kit
应用
细胞凋亡
储存与运输
储存条件
2-8°C储存,避光
运输条件
低温运输
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
输入批号以搜索分析图谱:
通过匹配包装上的批号来查找并下载产品的 COA，每批产品都进行了严格的验证，您可放心使用！
| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 B2518375 |
分析证书 | A598354 |
技术文档和文章
溶液计算器
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