Caspase 3/7 细胞凋亡检测试剂盒 (绿色荧光), 是通过Caspase 3/7底物 (绿色) 来检测活细胞中Caspase 3/7 的活化水平, 可快速、直观地评估细胞凋亡进程。Caspase 3/7底物为可穿透细胞膜的DEVD偶联荧光探针，在Caspase 3/7激活后被剪切并释放高亲和力DNA染料，将细胞核染成亮绿色。因此，Caspase 3/7底物 (绿色) 具有双功能，可检测细胞内Caspase 3活性并可视化细胞凋亡过程中的核形态变化。该产品适用于流式细胞仪、荧光酶标仪及荧光显微镜的多平台检测。
Caspase 3 (又称 CPP32/Apopain) 是细胞凋亡执行阶段的关键效应蛋白酶，其活性升高标志着凋亡程序的启动。本产品利用Caspase 3/7对底物序列Asp-Glu-Val-Asp (DEVD) 的高度特异性，将DEVD偶联至一种可穿透细胞膜的DNA荧光染料。该探针本身无荧光且因电荷排斥不与DNA结合；当细胞进入凋亡，活化的Caspase 3/7剪切探针，释放出DNA染料并与其结合，产生Ex/Em 488 nm/525 nm的明亮的绿色荧光。荧光强度与Caspase 3/7活性成正比，从而实现对细胞凋亡水平的定量或半定量分析。
该试剂盒包含 Caspase 3/7绿色荧光底物和 Caspase 3抑制剂 Ac-DEVD-CHO。每个试剂盒的检测数量基于100 μL检测体积中的5 μM底物浓度；实际检测次数可能因使用的浓度和染色体积而异。
产品组分
C1529092 | 组分名称 | 物理外观 | 50T | 100T | 储存条件 | 单次测试使用量 |
C1529092A | Caspase 3/7底物 (绿色) | 浅红色液体 | 25 μL | 50 μL | -20℃. 避光 | 0.5 μL/100 μL样品 |
C1529092B | Ac-DEVD-CHO | 无色液体 | 10 μL | 20 μL | -20℃. 避光 | 0.2 μL/100 μL样品 |
使用说明
一. 实验前准备
1. 试剂准备
Caspase 3/7底物 (绿色)：1 mM，即用型，避光保存，稀释后不可保存；
Ac-DEVD-CHO：10 mM，即用型，避光保存，稀释后不可保存。
2. 仪器准备
流式细胞仪：激发/发射波长 488/525 nm
荧光显微镜：激发/发射波长 488/525 nm
荧光酶标仪：激发/发射波长 488/525 nm
3. 实验设计
本试剂盒Caspase 3/7底物推荐使用浓度为5 μM, Ac-DEVD-CHO推荐浓度为10 μM。首次操作前，建议根据细胞类型对底物浓度、抑制剂浓度及凋亡诱导条件进行优化。
a. Caspase 3/7底物 (绿色) 的最佳浓度可以在1-10μM之间适当摸索。可以在实验开始或结束时直接加在细胞培养液、PBS或其它适当溶液中。对于贴壁细胞，在加底物前最好更换培养液。底物孵育结束后，培养液可自行选择是否更换。
b. Ac-DEVD-CHO是一种可逆抑制剂，可以抑制Caspase 3/7的活力, 工作浓度一般是底物的2-4倍。可预先加入培养液中处理细胞，室温孵育15-30分钟后再加入底物；也可以在诱导凋亡时就加入抑制剂，添加底物前换液时补加原来浓度的抑制剂。有一些细胞需要添加不可逆的抑制剂，如Z-DEVD-FMK, 可更有效地抑制 caspase 活性。
c. 各类型细胞株的凋亡诱导条件请参考文献。
4. 对照设置
建议设定以下对照：
组别 | Caspase 3/7底物 (绿色) | Ac-DEVD-CHO | 样品细胞 |
阴性对照 | + | - | 未诱导凋亡 |
阳性对照 | + | - | 凋亡诱导 |
阳性对照 | + | + | 凋亡诱导 |
二. 操作步骤
1. 流式细胞仪分析
(1) 选择合适的方法诱导细胞凋亡，未经诱导处理的作为阴性对照。抑制剂组添加一定浓度的抑制剂。
(2) 贴壁细胞先用胰酶或者其它细胞解离方法消化细胞，悬浮细胞直接收集细胞，离心，400×g离心5 min，去上清。
(3) 用细胞培养基或者PBS重悬细胞，调整细胞浓度为10⁶/mL。
(4) 吸取100 μL加入流式细胞检测板，加入0.5 μL的底物，立即混匀使底物终浓度为5 μM。不同的细胞合适的底物浓度不同，需要自行进行优化。
(5) 室温避光孵育15-30 min (如有需要可以孵育更长时间)。
(6) 孵育结束后30 min内用流式细胞仪 (最大激发/发射波长：488/525 nm) 分析。
2. 荧光显微镜分析
A. 对于悬浮细胞：
(1) 按照流式细胞仪分析步骤制样。
(2) 将染色好的细胞悬浮置于玻片上，用盖玻片盖住细胞。尽快使用适当的滤镜通过荧光显微镜分析细胞。
B. 对于贴壁细胞，按照以下操作步骤（以24孔板为例）：
(1) 细胞接种于爬片或腔室载玻片上，培养细胞。
(2) 通过所需方法诱导细胞凋亡，同时设置无诱导的对照组。
(3) 除去培养基，用PBS洗涤细胞2次。
(4) 准备工作液：每200 µL PBS添加1 µL Caspase3/7底物 (绿色)，轻柔混匀。
(5) 在每孔中加入200µL配好的工作液，室温避光孵育15-30 min。
(6) 使用合适的滤光片在荧光显微镜下分析细胞
3. 荧光酶标仪分析
(1) 贴壁细胞接种于黑色96孔板中，对于悬浮细胞，将密度调整至10⁶/mL，200 μL/孔。
(2) 选择合适的方法诱导细胞凋亡，未经诱导处理的作为阴性对照。抑制剂组添加一定浓度的抑制剂。
(3) 凋亡处理后，贴壁细胞用含5 μM底物的新鲜培养基或者PBS换液，Ac-DEVD-CHO抑制剂组要补充原来浓度的抑制剂。如果是悬浮细胞，则直接加入底物或抑制剂。
(4) 室温避光孵育15-30 min (如有需要可以孵育更长时间)。
(5) 对于悬浮细胞，轻轻摇晃重悬细胞。酶标仪设置激发波长488nm，发射波长525nm。贴壁细胞建议使用底读的方式。细胞密度的变化可能导致不准确的读数。
注意事项
1. 荧光染料有一定的淬灭性，请注意避免长时间在荧光显微镜下观察。
2. 本试剂盒组分须尽量避免反复冻融。如果多次使用，请注意适当分装。
3. Ac-DEVD-CHO在4ºC、冰浴等较低温度情况下会凝固而粘在离心管管底、管壁或管盖内，可以20-25ºC温育片刻至全部融解后使用。
4. 本试剂盒染色兼容多聚甲醛或甲醛固定，但与甲醇固定不兼容。建议用4%多聚甲醛固定10-15分钟，固定后的细胞可以用免疫染色通透液(Triton X-100) (I743383)通透处理后进行后续染色。过度固定会导致信号减弱。
5. 本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。
6. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
Caspase 3/7 Activity Apoptosis Assay Kit (Green, Fluorometric Method) detects the activation level of Caspase 3/7 in live cells via a green‑fluorescent Caspase 3/7 substrate, enabling rapid and intuitive evaluation of the progression of cellular apoptosis. The Caspase 3/7 substrate is a cell‑permeable DEVD‑conjugated fluorescent probe. Upon activation of Caspase 3/7, the substrate is cleaved to release a high‑affinity DNA dye that stains cell nuclei bright green. Therefore, the green‑fluorescent Caspase 3/7 substrate is bifunctional: it permits detection of intracellular Caspase 3 activity and visualization of nuclear morphological changes during apoptosis. This product is compatible with multi‑platform detection, including flow cytometry, fluorescence microplate readers and fluorescence microscopes.
Caspase 3 (also known as CPP32/Apopain) is a key effector protease in the execution phase of apoptosis. Elevated Caspase 3 activity marks the initiation of the apoptotic program. Taking advantage of the high specificity of Caspase 3/7 toward the substrate sequence Asp‑Glu‑Val‑Asp (DEVD), this kit conjugates DEVD to a cell‑permeable DNA fluorescent dye. The probe itself is non‑fluorescent and cannot bind DNA due to charge repulsion. When cells undergo apoptosis, activated Caspase 3/7 cleaves the probe and liberates the DNA dye, which then binds to DNA and generates bright green fluorescence with Ex/Em of 488 nm/525 nm. Fluorescence intensity is proportional to Caspase 3/7 activity, allowing quantitative or semi‑quantitative analysis of cellular apoptosis levels.
The kit contains green‑fluorescent Caspase 3/7 substrate and the Caspase 3 inhibitor Ac‑DEVD‑CHO. The number of tests per kit is calculated based on a substrate final concentration of 5 μM in a 100 μL reaction volume; the actual number of usable assays may vary depending on the applied substrate concentration and staining volume.
Components
C1529092 | Component Name | Appearance | 50T | 100T | Storage | Quantity Per Test | C1529092A | Caspase 3/7 substrate (Green) | Light red liquid | 25 μL | 50 μL | -20℃. Store in the dark. | 0.5 μL/100 μL sample | C1529092B | Ac-DEVD-CHO | Colorless liquid | 10 μL | 20 μL | -20℃. Store in the dark. | 0.2 μL/100 μL sample |
|
Instruction for use
I. Pre‑experiment Preparation
1. Reagent Preparation
Caspase 3/7 Substrate (Green): 1 mM, Ready‑to‑use; store protected from light. Do not store after dilution.
Ac‑DEVD‑CHO: 10 mM, Ready‑to‑use; store protected from light. Do not store after dilution.
2. Instrument Preparation
Flow cytometer: Excitation / Emission wavelength 488 nm/525 nm
Fluorescence microscope: Excitation / Emission wavelength 488 nm/525 nm
Fluorescence microplate reader: Excitation / Emission wavelength 488 nm/525 nm
3. Experimental Design
The recommended working concentration of Caspase 3/7 Substrate (Green) for this kit is 5 μM, and the recommended working concentration of Ac‑DEVD‑CHO is 10 μM. Prior to the first assay, optimization of substrate concentration, inhibitor concentration and apoptosis‑inducing conditions is suggested according to cell types.
a. The optimal concentration of Caspase 3/7 Substrate (Green) should be explored within the range of 1‑10 μM. The substrate can be added directly into cell culture medium, PBS or other appropriate solutions at the beginning or end of the experiment. For adherent cells, it is preferable to replace the culture medium before adding the substrate. Medium replacement is optional after substrate incubation.
b. Ac‑DEVD‑CHO is a reversible inhibitor that suppresses Caspase 3/7 activity. Its working concentration is generally 2‑4‑fold that of the substrate. Cells can be pre‑treated with the inhibitor in culture medium and incubated at room temperature for 15‑30 min prior to substrate addition. Alternatively, the inhibitor can be added at the onset of apoptosis induction; replenish the inhibitor to the original concentration during medium exchange before adding the substrate. For certain cell lines, irreversible inhibitors such as Z‑DEVD‑FMK may be required for more effective suppression of caspase activity.
c. Refer to relevant literature for apoptosis‑inducing conditions of various cell lines.
4. Control Setup
Recommended controls are set as follows:
Group | Caspase 3/7 Substrate (Green) | Ac‑DEVD‑CHO | Sample Cells | Negative Control | + | － | Non‑apoptosis‑induced | Positive Control | + | － | Apoptosis‑induced | Positive Control | + | + | Apoptosis‑induced |
|
|
Ⅱ. Operating Procedures
1. Flow Cytometry Analysis
(1) Induce cellular apoptosis using an appropriate method. Use untreated, non‑induced cells as the negative control. Add the designated concentration of inhibitor to the inhibitor control group.
(2) For adherent cells, dissociate cells with trypsin or other cell‑dissociation reagents. Collect suspension cells directly. Centrifuge at 400 × g for 5 min and discard the supernatant.
(3) Resuspend cells in cell culture medium or PBS and adjust the cell density to 10⁶ cells/mL.
(4) Transfer 100 μL of cell suspension into a flow cytometry plate. Add 0.5 μL substrate and mix immediately to reach a final substrate concentration of 5 μM. Optimal substrate concentration varies among cell types and should be optimized empirically.
(5) Incubate for 15‑30 min at room temperature protected from light (longer incubation time may be applied if necessary).
(6) Perform analysis within 30 min after incubation using a flow cytometer (Ex/Em: 488 nm / 525 nm).
2. Fluorescence Microscopy Analysis
A. Suspension Cells
(1) Prepare samples following the sample‑preparation steps described for flow cytometry analysis.
(2) Place stained cell suspension onto a glass slide and cover with a coverslip. Analyze cells promptly under a fluorescence microscope with appropriate filter sets.
B. Adherent Cells (24‑well plate as example)
(1) Seed cells onto glass coverslips or chamber slides for culture.
(2) Induce apoptosis with the desired treatment; set up a non‑induced control group in parallel.
(3) Remove culture medium and wash cells twice with PBS.
(4) Prepare working solution: add 1 μL Caspase 3/7 Substrate (Green) per 200 μL PBS and mix gently.
(5) Add 200 μL prepared working solution to each well, and incubate at room temperature for 15‑30 min protected from light.
(6) Analyze cells under a fluorescence microscope using suitable filter sets.
3. Fluorescence Microplate Reader Analysis
(1) Seed adherent cells in black 96‑well plates. For suspension cells, adjust cell density to 10⁶ cells/mL and add 200 μL per well.
(2) Induce cellular apoptosis by an appropriate method. Use non‑treated cells as the negative control. Add the designated concentration of inhibitor to the inhibitor group.
(3) After apoptosis induction, replace medium of adherent cells with fresh medium or PBS containing 5 μM substrate. For the Ac‑DEVD‑CHO inhibitor group, replenish inhibitor to its original working concentration. For suspension cells, add substrate or inhibitor directly.
(4) Incubate for 15‑30 min at room temperature protected from light (longer incubation time may be applied if necessary).
(5) Gently agitate to resuspend suspension cells. Set the microplate reader to excitation 488 nm and emission 525 nm. Bottom‑reading mode is recommended for adherent cells. Variation in cell density may cause inaccurate readings
Precautions
1. Fluorescent dyes are susceptible to photobleaching. Avoid prolonged observation under the fluorescence microscope.
2. Repeated freeze‑thaw cycles of kit components should be avoided. For frequent usage, aliquot the reagents appropriately.
3. Ac‑DEVD‑CHO may solidify at low temperatures such as 4 °C or on ice, adhering to the bottom, wall or cap of centrifuge tubes. Warm the tube briefly at 20‑25 °C until it is completely dissolved before use.
4. Staining with this kit is compatible with paraformaldehyde/formaldehyde fixation, but incompatible with methanol fixation. Fix cells with 4% paraformaldehyde for 10‑15 min. Fixed cells can be permeabilized with Immunostaining Permeabilization Buffer with Triton X-100 (Cat. No. I743383) for subsequent staining. Over‑fixation will attenuate fluorescence signal.
5. This product is for research use only by qualified laboratory personnel. It shall not be used for clinical diagnosis or therapy, food or pharmaceutical applications, and shall not be stored in residential premises.
6. For personal safety and health, wear a lab coat and disposable gloves during operation
危险品化学品经营许可证（带存储）