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概述
aladdin 488 Caspase-3活细胞分析试剂盒包含aladdin 488 Caspase-3底物和Ac-DEVD-CHO Caspase-3抑制剂。aladdin 488 Caspase-3 Substrate为基于Caspase-3活力检测细胞凋亡提供了有效的工具，适用于荧光显微术和流式细胞术。相比其他基于（FLICA）分析的Caspase的荧光底物或荧光抑制剂，aladdin 488 Caspase-3 Substrate检测Caspase-3活性的同时不会抑制完整细胞的凋亡过程。Substrate由耦合Caspase-3 DEVD识别序列的荧光DNA 染料组成。Substrate最初无荧光，穿过细胞膜进入细胞质。在凋亡细胞中，Caspase-3剪切Substrate，释放高亲和性的DNA染色，这种染料迁移到细胞核标记DNA并发出明亮的绿色荧光。因此，aladdin 488 Caspase-3 Substrate是双功能的，既可以检测Caspase-3活性，又可视化细胞核在细胞凋亡进行中的形态学变化。aladdin 488染色可以甲醛固定并兼容后续免疫染色实验。
产品参数：
aladdin 488：Ex/Em = 500/530 nm (with DNA)
组分：
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. 细胞可以用终浓度为1 μM的Hoechst 33342染料共同染色，使细胞核产生蓝色荧光染色（Ex/Em = 346/460 nm）。
3. aladdin 488 染色可以被甲醛固定，但与甲醇固定不兼容。
4. 甲醛固定的aladdin 488染色细胞可以用0.1% TritonX-100处理后进行后续染色，但处理后的染色的亮度可能会减弱。
5. 荧光染料均存在淬灭问题，请尽量注意避光，以减缓荧光淬灭。
6. 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：
Caspase 3 kit、细胞凋亡检测
使用方法：
1. 实验优化 下面提供的实验步骤根据终点法检测制度。 aladdin 488 Substrate 也可以进行细胞长时间孵育课程研究。细胞密度、 底物浓度和抑制剂浓度可能需要优化。最佳底物浓度可能在 1-10 μM 之间。细胞可以在培养基、PBS 或其他您所选择的缓冲 液中孵育底物。对于贴壁细胞，我们建议更换新鲜含有底物的培养基，以防背景的不均一性。底物孵育后换液或洗涤细胞的 操作是自由选择的。
2. 对照 我们建议您设定以下对照：
A. 阴性对照：不诱导凋亡的细胞；
B. 阳性对照：诱导凋亡的细胞；
C. 抑制剂对照：诱导细胞凋亡同时（或提前 10-30 min）孵育 Caspase-3/7 抑制剂，最后再添加 aladdin 488 Caspase-3 底 物。
3. Ac-DEVD-CHO Caspase-3 抑制剂对照 试剂盒中的 Caspase-3/7 抑制剂 Ac-DEVD-CHO 可以用来确认 Caspase-3/7 依赖于 aladdin 488 的荧光信号。对于抑制 剂对照，抑制剂的终浓度应该至少是底物浓度的 2 倍（例如当使用 5 μM aladdin 488 底物时，Ac-DEVD-CHO 浓度为 10 μM）。添加底物前在室温下孵育 Ac-DEVD-CHO 15-30 min，加入底物后，孵育液中继续保留抑制剂。Ac-DEVD-CHO 是可 逆的竞争性抑制剂。在某些细胞类型，有效的 Caspase-3/7 抑制剂需要使用不可逆的抑制剂，如 Z-DEVD-FMK，或需要在凋 亡诱导之前或诱导过程中添加抑制剂。
4. 流式细胞术
（1）选择合适的方法诱导细胞凋亡，未经处理的细胞样本作为对照。
（2）贴壁细胞，执行 aladdin 488 Caspase-3 实验前先用胰蛋白酶或其他方法消化细胞。
（3）用培养基或缓冲液重悬细胞，使细胞密度为 106个/mL
（4）吸取 0.2 mL 细胞悬液至流式细胞试管。
（5）抑制剂对照样本，用 Ac-DEVD-CHO 处理细胞（见上文 3. Ac-DEVD-CHO Caspase-3 抑制剂对照）。
（6）200 μL 细胞悬液中加入 5 μL 0.2 mM 的 Substrate 并立即混匀使底物浓度为 5 μM。不同细胞的最佳底物浓度可能不同， 需分析优化。
（7）室温避光孵育细胞 15-30 min。
（8）加入 300 μL 培养基或 PBS，流式细胞仪分析。检测绿色荧光的通道（Ex/Em = 485/515 nm）。
5. 荧光显微镜
（1）选择合适的方法诱导细胞凋亡，未经处理的细胞样本作为对照。
（2）抑制剂对照样本，用 Ac-DEVD-CHO 处理细胞（见上文 3. Ac-DEVD-CHO Caspase-3 抑制剂对照）。
（3）用含有 5 μM Substrate 的新鲜培养基或 PBS 对细胞进行换液（见上文 1. 实验优化）。对于抑制剂对照组，抑制剂与底 物一同孵育。
（4）室温下孵育细胞 30 min 或更长时间。
（5）细胞可以在含有 Substrate 的培养基中直接观察。对于终点分析法，PBS 清洗细胞，荧光显微镜观察细胞，使用观察绿 色荧光的滤片（Ex/Em = 485/515 nm）。
6. 荧光酶标仪
（1）贴壁细胞生长在黑色 96 孔板中；悬浮细胞，调整密至 106个/mL，0.2 mL 细胞悬液分装到一孔。
（2）选择合适的方法诱导细胞凋亡，未经处理的细胞样本作为对照。 注：细胞可能在管或瓶中处理，然后转移到 96 孔检测板。
（3）抑制剂对照样本，用 Ac-DEVD-CHO 处理细胞（见上文 3. Ac-DEVD-CHO Caspase-3 抑制剂对照）。
（4）对于悬浮细胞，直接添加 Substrate 混匀。对于贴壁细胞，用含有 5 μM Substrate 的新鲜培养基或 PBS 对细胞进行换液 （见上文 1. 实验优化）。对于抑制剂对照组，抑制剂与底物一同孵育。
（5）细胞可以在含有 Substrate 的培养基中直接观察。
（6）对于悬浮细胞，轻轻摇晃重悬细胞。荧光酶标仪设置激发波长 488 nm 和发射波长 520 nm。建议贴壁细胞使用底部采 集方式。贴壁细胞密度的变化可能导致不准确的读数。
The aladdin 488 Caspase-3 live cell assay kit contains the aladdin 488 Caspase-3 substrate and the Ac-DEVD-CHO Caspase-3 inhibitor. aladdin 488 Caspase-3 Substrate provides an effective tool for detecting apoptosis based on Caspase-3 activity, suitable for fluorescence microscopy and flow cytometry. Compared with other fluorescent substrates or fluorescent inhibitors of Caspase based on ( FLICA ) analysis, aladdin 488 Caspase-3 Substrate does not inhibit the apoptosis process of intact cells while detecting Caspase-3 activity. Substrate is composed of fluorescent DNA dyes coupled with Caspase-3 DEVD recognition sequence. Substrate initially had no fluorescence and entered the cytoplasm through the cell membrane. In apoptotic cells, Caspase-3 cleaves the Substrate and releases high-affinity DNA staining, which migrates to the nucleus to label DNA and emits bright green fluorescence.Therefore, aladdin 488 Caspase-3 Substrate is bifunctional, which can not only detect Caspase-3 activity, but also visualize the morphological changes of the nucleus during apoptosis. Aladdin 488 staining can be fixed in formaldehyde and compatible with subsequent immunostaining experiments.
Parameters:
aladdin 488：Ex/Em = 500/530 nm (with DNA)
Component：
Points for attention：
1.Please instantaneously centrifuge the product to the bottom of the tube before use, and then carry out subsequent experiments.
2.Cells can be co-stained with a final concentration of 1μM Hoechst 33342 dye to produce blue fluorescence staining of the nucleus ( Ex / Em = 346 / 460 nm ).
3.Aladdin 488 staining can be fixed by formaldehyde, but it is not compatible with methanol fixation.
4.Formaldehyde-fixed aladdin 488-stained cells can be treated with 0.1 % TritonX-100 for subsequent staining, but the brightness of the treated staining may be weakened. 5.Fluorescent dyes all have quenching problems, please try to avoid light to slow down the fluorescence quenching.
6.For your safety and health, please wear experimental clothes and wear disposable gloves.
Scope of application：
Caspase 3 kit and apoptosis detection
Usage:
1. Experimental optimization: The experimental steps provided below are based on the endpoint detection system. Aladdin 488 Substrate can also be used for long-term cell incubation course research. Cell density, substrate concentration, and inhibitor concentration may need to be optimized. The optimal substrate concentration may be between 1-10 μ Between M. Cells can be incubated with substrates in culture medium, PBS, or other buffer of your choice. For adherent cells, we recommend replacing them with fresh culture media containing substrates to prevent background heterogeneity. The operation of changing the medium or washing the cells after substrate incubation is freely selectable.
2. We suggest that you set the following controls:
A. Negative control: cells that do not induce apoptosis;
B. Positive control: cells that induce apoptosis;
C. Inhibitor control: Induce cell apoptosis while incubating Caspase-3/7 inhibitors (or 10-30 minutes in advance), and finally add Aladdin 488 Caspase-3 substrate.
3. The Caspase-3/7 inhibitor Ac-DEVD-CHO in the Ac-DEVD-CHO Caspase-3 inhibitor control kit can be used to confirm that Caspase-3/7 depends on the fluorescence signal of aladdin 488. For inhibitor control, the final concentration of the inhibitor should be at least twice the substrate concentration (e.g. when using 5 μ At substrate M aladdin 488, the concentration of Ac-DEVD-CHO is 10 μ M) . Before adding the substrate, incubate Ac-DEVD-CHO at room temperature for 15-30 minutes. After adding the substrate, continue to retain the inhibitor in the incubation solution. Ac-DEVD-CHO is a reversible competitive inhibitor. In certain cell types, effective Caspase-3/7 inhibitors require the use of irreversible inhibitors, such as Z-DEVD-FMK, or the addition of inhibitors before or during apoptosis induction.
4. Flow cytometry
(1) Choose appropriate methods to induce cell apoptosis, with untreated cell samples as controls.
(2) Adhering cells should be digested with trypsin or other methods before performing the aladdin 488 Caspase-3 experiment.
(3) Resuspend cells with culture medium or buffer to achieve a cell density of 106 cells/mL
(4) Suck 0.2 mL of cell suspension into a flow cytometry test tube.
(5) Inhibitor control samples were treated with Ac-DEVD-CHO on cells (see 3 above) Ac-DEVD-CHO Caspase-3 inhibitor control.
(6) 200 μ Add 5 to L cell suspension μ Substrate of 0.2 mM and immediately mix to achieve a substrate concentration of 5 μ M. The optimal substrate concentration for different cells may vary and requires analysis and optimization.
(7) Incubate cells at room temperature in dark for 15-30 minutes.
(8) Join 300 μ L-medium or PBS, analyzed by flow cytometry. Detect the channel for green fluorescence (Ex/Em=485/515 nm).
5. Fluorescence microscope
(1) Choose appropriate methods to induce cell apoptosis, with untreated cell samples as controls.
(2) Inhibitor control samples were treated with Ac-DEVD-CHO on cells (see 3 above) Ac-DEVD-CHO Caspase-3 inhibitor control.
(3) Using a solution containing 5 μ M Substrate's fresh culture medium or PBS is used to replace the cell culture medium (see 1 above) Experimental optimization). For the inhibitor control group, the inhibitor was incubated together with the substrate.
(4) Incubate cells at room temperature for 30 minutes or longer.
(5) Cells can be directly observed in culture media containing Substrate. For the endpoint analysis method, PBS was used to clean the cells, fluorescence microscopy was used to observe the cells, and a filter (Ex/Em=485/515 nm) was used to observe green fluorescence.
6. Fluorescence enzyme-linked immunosorbent assay (ELISA) reader
(1) Adherent cells grow in black 96 well plates; Suspend cells, adjust the density to 106 cells/mL, and divide 0.2 mL of cell suspension into one well.
(2) Choose appropriate methods to induce cell apoptosis, with untreated cell samples as controls. Note: Cells may be processed in tubes or bottles and then transferred to a 96 well detection plate.
(3) Inhibitor control samples were treated with Ac-DEVD-CHO on cells (see 3 above) Ac-DEVD-CHO Caspase-3 inhibitor control.
(4) For suspended cells, directly add Substrate and mix well. For adherent cells, use a solution containing 5 μ M Substrate's fresh culture medium or PBS is used to replace the cell culture medium (see 1 above) Experimental optimization). For the inhibitor control group, the inhibitor was incubated together with the substrate.
(5) Cells can be directly observed in culture media containing Substrate.
(6) For suspended cells, gently shake to resuspend the cells. The fluorescence enzyme-linked immunosorbent assay instrument is set with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. Suggest using bottom collection method for adherent cells. Changes in the density of adherent cells may lead to inaccurate readings.
规格
英文名称
aladdin™ 488 caspase-3 live cell assay kit
储存与运输
储存条件
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低温运输
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