基本描述

规格或纯度

BioReagent, 生物染色剂, 用于显微镜, 适用于免疫荧光(IF), 即用型

稳定性与储存

Store at 2-8°C long term (12 months). Store in the dark. Do not freeze.

英文名称

Annexin V-Biotin/PI Apoptosis Detection Kit

储存温度

2-8°C储存,避光,禁止冷冻

运输条件

冰袋运输

产品介绍

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。

产品组分

A1373480	Component	20 T	50 T	100 T	Storage	Quantity Per Test
A1373480A	10x Annexin V Binding Buffer	5 mL	10 mL	20 mL	2-8℃.	200 μL 0.5-1.0x10^5 cells
A1373480B	Annexin V-Biotin	450 μL	1.5 mL	2.5 mL	2-8℃, Store in the dark.	20 μL 0.5-1.0x10^5 cells
A1373480C	Propidium iodide Staining Solution (PI)	15 μL	30 μL	55 μL	2-8℃, Store in the dark.	0.5 μL 0.5-1.0x10^5 cells
A1373480D	Streptavidin protein (FITC)	10 μL	15 μL	25 μL	2-8℃, Store in the dark.	0.2 μL 0.5-1.0x10^5 cells

注: 每个Test推荐染色的细胞数量为 0.5-1.0x10^5 cells.

使用方法

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0.5-1.0x10^6 /mL 的密度用1x Annexin V结合缓冲液重悬；

3. 每200 μL细胞悬液中加入20 μL 荧光标记Annexin V-Biotin及0.5 μL PI染料；

4. 轻柔涡旋混匀细胞，室温避光孵育10 min;

5. 400g，离心5 min，去除上清;

6. Streptavidin protein (FITC)用1x Annexin V结合缓冲液1:1000稀释;

7. 加入200 μL稀释好的Streptavidin protein (FITC)，轻柔重悬细胞;

8. 4℃避光孵育30 min;

9. 400g，离心5 min去除Streptavidin protein (FITC);

10. 加入200 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. 碘化丙啶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.

Kit Contents

A1373480	Component	20 T	50 T	100 T	Storage	Quantity Per Test
A1373480A	10x Annexin V Binding Buffer	5 mL	10 mL	20 mL	2-8℃.	200 μL 0.5-1.0x10^5 cells
A1373480B	Annexin V-Biotin	450 μL	1.5 mL	2.5 mL	2-8℃, Store in the dark.	20 μL 0.5-1.0x10^5 cells
A1373480C	Propidium iodide Staining Solution (PI)	15 μL	30 μL	55 μL	2-8℃, Store in the dark.	0.5 μL 0.5-1.0x10^5 cells
A1373480D	Streptavidin protein (FITC)	10 μL	15 μL	25 μL	2-8℃, Store in the dark.	0.2 μL 0.5-1.0x10^5 cells

Note: The recommended number of cells to stain per test is 0.5-1.0x10^5 cells.

Instruction for use

1. Before use, dilute the 10x binding buffer with deionized water to 1x (1 mL of 10x buffer plus 9 mL of deionized water);

2. After pre-cooling PBS and washing the cells twice, resuspend them in 1x Annexin V binding buffer at a density of 0.5-1.0x10^6 /mL;

3. Add 20 μL of fluorescently labeled Annexin V-Biotin and 0.5 μL of PI dye to each 200 μL cell suspension;

4. Gently vortex the cells and incubate at room temperature in the dark for 10 minutes;

5. Centrifuge at 400g for 5 minutes, remove the supernatant;

6. Dilute Streptavidin protein (FITC) with 1x Annexin V binding buffer at a ratio of 1:1000;

7. Add 200 μL of the diluted Streptavidin protein (FITC) and gently resuspend the cells;

8. Incubate at 4°C in the dark for 30 minutes;

9. Centrifuge at 400g for 5 minutes to remove the Streptavidin protein (FITC);

10. Add 200 μL of the binding buffer and complete the instrument analysis within 1 hour.

Matters needing attention

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.

图片

Flow Cytometry: Annexin V-Biotin/PI Apoptosis Detection Kit (A1373480)

Annexin V-Biotin/PI Apoptosis Detection Kit (A1373480) - Flow Cytometry
Flow cytometric analysis was performed on Jurkat cells, either untreated (left panel) or treated with 2 µM camptothecin (C111281) for 16 hours (right panel), using the Annexin V-Biotin/PI Apoptosis Detection Kit (A1373480).
The cells were stained with Annexin V-Biotin and PI for 10 minutes, centrifuged, and the supernatant was removed. After resuspension in Streptavidin protein (FITC), the cells were incubated at 4 °C for 30 minutes and then analyzed. In the untreated sample (left panel), the majority of cells were negative for both Annexin V-Biotin and PI, indicating a viable, non-apoptotic population. Following the 16-hour treatment with camptothecin (right panel), three main cell populations were identified:
1. Viable, non-apoptotic cells: Annexin V-Biotin negative/PI negative.
2. Early apoptotic cells: Annexin V-Biotin positive/PI negative.
3. Late apoptotic and dead cells: Annexin V-Biotin positive/PI positive.

名称和识别符

分子类型	试剂盒

化学和物理性质

敏感性	Light-sensitive

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:

溶液计算器

摩尔浓度计算器

计算溶液所需的质量、体积或浓度。

质量

=

浓度

x

体积

x

分子量

g/mol

稀释计算器

计算配制储备溶液所需的稀释度。

浓度1（C1）

x

体积 1 (V1)

=

浓度 2 (C2)

x

体积 2 (V2)

复溶计算器

复溶计算器允许您快速计算试剂的体积，以复溶小瓶。只需输入试剂的质量和目标浓度，计算器将确定其余部分。

体积（添加到小瓶中）

=

质量（小瓶）

÷

所需的复溶浓度

货号 (SKU)	包装规格	是否现货
A1373480-20T	20T	期货
A1373480-50T	50T	期货
A1373480-100T	100T	期货

Annexin V-Biotin/PI细胞凋亡检测试剂盒

库存信息

库存信息

库存信息

基本描述

图片

名称和识别符

化学和物理性质

质检证书（CoA,COO,BSE/TSE 和分析图谱)

溶液计算器

摩尔浓度计算器

稀释计算器

复溶计算器