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。PI是一种荧光核染料，对活细胞和凋亡细胞不敏感，但对死细胞有红色荧光染色，与细胞内的核酸结合紧密。
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。在提供的结合缓冲液中，用Annexin V-Biotin和PI染色细胞群后，就可以将处于不同凋亡时期的细胞区分开来。此外，本试剂盒含有凋亡诱导剂，可以诱导细胞到凋亡状态，标准化凋亡检测和结果。10-15分钟即可染色，方便快捷，分群清晰，结果可靠。
产品组分
A1524968 | 组分名称 | 物料外观 | 20T | 50T | 100T | 储存 | 单次测试使用量 | A1524968A | 10× Annexin V 结合缓冲液 | 无色透明液体 | 5 mL | 10 mL | 20 mL | 2-8℃ | 200 μL/0.5-1.0×10⁵细胞 | A1524968B | Annexin V-Biotin | 无色透明液体 | 0.45 mL | 1.5 mL | 2.5 mL | 2-8℃, 避光. | 20 μL/0.5-1.0×10⁵细胞 | A1524968C | PI染色液 | 橙红色液体 | 40 μL | 100 μL | 200 μL | 2-8℃, 避光. | 2 μL/0.5-1.0×10⁵细胞 | A1524968D | FITC标记的链霉亲和素 (100×) | 黄色透明液体 | 40 μL | 100 μL | 200 μL | 2-8℃, 避光. | 2 μL/0.5-1.0×10⁵细胞 | A1524968E | 凋亡诱导剂 (1mM) | 浅黄色液体 | 20 μL | 50 μL | 100 μL | 2-8℃, 避光. | 1 μL/0.5-1.0×10⁶细胞 |
注: 单次测试推荐染色的细胞数量为 0.5-1.0×10⁵细胞; 组分E收到后建议分装保存于-20℃, 避免反复冻融. |
试剂准备
1× Annexin V结合缓冲液：临用前配制，用去离子水把10× Annexin V结合缓冲液稀释成1×。
Annexin V-Biotin：即用型；使用前，平衡到室温；未用完的试剂分装后2-8℃避光保存。
PI染色液：即用型；使用前，平衡到室温；未用完的试剂分装后2-8℃避光保存。
FITC标记的链霉亲和素 (100×)：临用前配制，用1× Annexin V结合缓冲液稀释成1×，配制好的试剂在2-8℃避光保存，一周内有效。
凋亡诱导剂 (1mM)：收到后建议分装，避光保存于-20℃，使用前，平衡到室温。
使用说明
1. 细胞凋亡阳性诱导
(1) 对于待诱导凋亡的培养细胞， 按照0.5-1.0×10⁶/mL细胞1-10 μM 的凋亡诱导剂浓度稀释到培养基中。
(2) 在4、8、12、16或24h后观察细胞凋亡的情况。通常16-24h后，在光学显微镜下可以看到明显的细胞形态的变化，此时可以用于细胞凋亡染色观察 (最佳诱导浓度及时间根据不同的细胞类型参考文献或进行预实验确定)。 比如诱导2×10⁶的Jurkat细胞凋亡，将细胞密度调整为1× 10⁶后，加入2 μM 凋亡诱导剂，37℃二氧化碳培养箱培养24h后可检测到明显的凋亡现象。
2. 细胞凋亡检测
(1) 将细胞收集到离心管内，1000 rpm左右离心5 min；
注：贴壁细胞使用胰酶（不含EDTA）消化后离心收集细胞，需控制胰酶消化时间，避免消化时间过长造成细胞膜结构损伤而出现细胞坏死的假阳性。
(2) 用移液器小心吸除上清，加入4℃预冷的1×PBS缓冲液重悬细胞，再次离心沉淀细胞，小心吸除上清；
(3) 每100 μL细胞悬液中加入20 μL Annexin V-Biotin及2 μL PI染色液；
(4) 轻柔涡旋混匀细胞，室温避光孵育10-15 min;
(5) 400×g离心5 min，去除上清;
(6) 加入200 μL稀释好的FITC标记的链霉亲和素，轻柔重悬细胞;
(7) 4℃避光孵育30 min;
(8) 400×g离心5 min去除FITC标记的链霉亲和素；
(9) 加入200 μL 1×Annexin V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. 由于细胞凋亡是一个快速的过程，建议样品在染色后1小时之内进行分析
4. 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实验中务必使用不含EDTA或其他钙螯合剂的缓冲液。
Annexins are a family of calcium-dependent phospholid-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 phospholid 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. PI is a fluorescent nuclear dye that does not stain viable cells or apoptotic cells, but selectively stains dead cells with red fluorescence by binding tightly to intracellular nucleic acids.
This kit is intended for the identification and enumeration of dead cells (e.g., apoptotic or necrotic cells) by flow cytometry. Staining with Annexin V-Biotin and PI enables the discrimination of cells at various apoptotic stages. In addition, this kit contains an apoptosis inducer that can drive cells into an apoptotic state, thereby standardizing apoptosis detection and results. Staining takes only 10-15 minutes, offering convenience and speed, with clear population segregation and reliable results.
Components
A1524968 | Component Name | Appearance | 20T | 50T | 100T | Storage | Quantity Per Test | A1524968A | 10× Annexin V Binding Buffer | Colourless liquid | 5 mL | 10 mL | 20 mL | 2-8℃ | 200 μL/0.5-1.0×10⁵ cells | A1524968B | Annexin V-Biotin | Colourless liquid | 0.45 mL | 1.5 mL | 2.5 mL | 2-8℃. Store in the dark. | 2 μL/0.5-1.0×10⁵ cells | A1524968C | PI Staining Solution | Orange Red liquid | 40 μL | 100 μL | 200 μL | 2-8℃. Store in the dark. | 2 μL/0.5-1.0×10⁵ cells | A1524968D | Streptavidin protein (FITC) (100×) | Yellow liquid | 40 μL | 100 μL | 200 μL | 2-8℃. Store in the dark. | 2 μL/0.5-1.0×10⁵ cells | A1524968E | Apoptosis Inducer | Pale yellow liquid | 20 μL | 50 μL | 100 μL | 2-8℃. Store in the dark. | 1 μL/0.5-1.0×10⁶ cells |
Note: The recommended number of cells to stain per test is 0.5-1.0×10⁵ cells. Component E should be aliquoted and stored at -20°C upon receipt; Avoid repeated freezing and thawing. |
Reagent Preparation
1× Annexin V Binding Buffer: Prepare immediately before use. Dilute the 10× Annexin V Binding Buffer with deionized water to 1×.
Annexin V‑PE: Ready‑to‑use; equilibrate to room temperature before use. Any unused reagent should be aliquoted and stored at 2-8 °C protected from light.
PI Staining Solution: Ready‑to‑use; equilibrate to room temperature before use. Any unused reagent should be aliquoted and stored at 2-8 °C protected from light.
Streptavidin protein (FITC) (100×): Prepare immediately before use. Dilute to 1× with 1× Annexin‑V Binding Buffer. The prepared working solution can be stored in the dark at 2-8 °C and is valid within one week.
Apoptosis Inducer (1mM): Upon receipt, it is recommended to aliquot and store at -20 °C protected from light. Before use, equilibrate to room temperature.
Instruction for use
1. Positive Induction of Apoptosis
(1) For cultured cells to be induced into apoptosis, adjust the cell density of 0.5-1.0×10⁶ cells/mL and dilute the apoptosis inducer into the culture medium to a final concentration of 1-10 μM.
(2) Observe the cells for apoptosis at 4, 8, 12, 16, or 24 h post‑induction. Typically, after 16-24 h, obvious morphological changes can be observed under a light microscope, at which point the cells are suitable for apoptosis staining and observation (the optimal induction concentration and time should be determined by referring to literature or performing preliminary experiments for different cell types). For example, to induce apoptosis of 2×10⁶ Jurkat cells, add 2 μL apoptosis inducer in 2 mL medium, and culture in a 37 °C CO₂ incubator for 24 h; significant apoptosis can then be detected.
2. Apoptosis Detection
(1) Collect cells into a centrifuge tube and centrifuge at approximately 1,000 rpm for 5 min.
Note: For adherent cells, use trypsin (without EDTA) to detach the cells, then centrifuge to collect them. The digestion time should be controlled to avoid over‑digestion, which may damage the cell membrane structure and cause false‑positive results for necrosis.
(2) Carefully aspirate the supernatant with a pipette, resuspend the cells in 1× PBS buffer pre‑cooled to 4 °C, centrifuge again to pellet the cells, and carefully remove the supernatant.
(3) To every 100 μL of cell suspension, add 20 μL of Annexin V‑Biotin and 2 μL of PI staining solution.
(4) Gently vortex to mix the cells, and incubate at room temperature for 10-15 min in the dark.
(5) Centrifuge at 400×g for 5 min and discard the supernatant.
(6) Add 200 μL of diluted FITC‑conjugated streptavidin and gently resuspend the cells.
(7) Incubate the samples at 4 °C in the dark for 30 min.
(8) Centrifuge at 400×g for 5 min to remove unbound FITC‑conjugated streptavidin.
(9) Add 200 μL of 1× Annexin V Binding Buffer, and perform flow‑cytometric analysis within 1 h.
Precautions
1. Protect from light during use to minimize fluorescence quenching.
2. PI dye is highly toxic and mutagenic; handle with care.
3. Since apoptosis is a rapid process, it is recommended to analyze the samples within 1 h after staining.
4. Because the binding of Annexin V to phosphatidylserine (PS) is calcium‑dependent, it is essential to use buffers that do not contain EDTA or other calcium chelators in Annexin V assays.
危险品化学品经营许可证（带存储）