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LumiDye™ 488 Caspase 3 底物 (1 mM)
有货
为什么选择此级别
BioReagent,适用于荧光分析 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
避光,-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
LumiDye™ 488 Caspase-3 Substrate基于caspase-3/7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，LumiDye™ 488 Caspase-3 Substrate检测caspase-3/7活性的同时不会抑制完整细胞的凋亡过程。
Substrate 由耦合 caspase-3/7 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-3/7 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，LumiDye™ 488 Caspase-3 Substrate 是双功能的，既可以检测caspase-3/7 活性，又可视化细胞核在细胞凋亡进行中的形态学变化。LumiDye™ 488染色可以甲醛固定并兼容后续免疫染色实验。
LumiDye™ 488 Caspase-3 Substrate 提供 DMSO 和PBS 两种溶解形式。PBS形式可用于对DMSO毒性敏感的细胞，在对DMSO不敏感的细胞类型中，添加 DMSO 溶解形式可增强 LumiDye™ 488的孵育染色效果。
产品特点：
⁕ 稳定性好：荧光亮度高且抗淬灭性好；
⁕ 批间差小：批间差稳定。
产品参数：
Ex/Em: 500/530 nm (with DNA)
自备材料：
1. 耗材：1.5 mL 离心管
2. 仪器：流式细胞仪
操作步骤：
1. 实验优化
下面提供的实验步骤根据终点法检测。LumiDye™ 488 Substrate也可以进行细胞长时间孵育实验研究。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 或其他所选择的缓冲液中孵育底物。对于贴壁细胞，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
建议设定以下对照：
(1) 阴性对照：不诱导凋亡的细胞。
(2) 阳性对照：诱导凋亡的细胞。
(3) 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LumiDye™ 488 Caspase 3底物。
Ac-DEVD-CHO Caspase 3抑制剂对照：
Caspase-3/7抑制剂Ac-DEVD-CHO (货号 A305146) 可以用来确认Caspase-3/7 依赖于 LumiDye™ 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 LumiDye™ 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。
3. 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 LumiDye™ 488 Caspase 3 实验前先用胰蛋白酶或其他方法消化细胞。
(3) 冲液重悬细胞，使细胞密度为10⁶个/mL。
(4) 取 0.2 mL 细胞悬液至流式细胞试管。
(5) 对照样本，用Ac-DEVD-CHO处理细胞 (见第2点，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)。
4. 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 (见第2点，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 (见第1点，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)。
5. 荧光酶标仪
(1) 贴壁细胞生长在黑色96孔板中；悬浮细胞，调整密度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 (见第2点，Ac-DEVD-CHO Caspase 3 抑制剂对照）。
(4) 对于悬浮细胞，直接添加 Substrate 混匀。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 (见第1点，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。
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. LumiDye™ 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 LumiDye™ 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. 本产品仅限于科研，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。
7. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
常见问题：
1. 问：产品稳定性如何？
答：该物质稳定性很好，用户反馈该产品37℃放置4~5天，效果仍然很好该物质稳定性很好。
2. 问：何时加入细胞中？
答：该物质实验前期、后期加入细胞均可，它不影响细胞凋亡进程，可实时监测 Caspase 3 活性。
3. 问：可用于组织染色吗？
答：本公司未进行活组织染色实验，有文献报道可用于胚胎组织或三维培养细胞。
4. 问：可用于随后的免疫染色吗？
答：可以。推荐使用2%～4%的多聚甲醛室温固定10~15 min，固定时间过长会导致信号下降。
5. 问：特异性如何？
答：与其它 Caspase 3 底物相似，LumiDye™ Caspase 3 Substrate 是基于能被 Caspase 7 切割的 DEVD Caspase 3 共同序列，其它的半胱天冬酶也可能因与底物特异性序列重叠而切割 DEVD 底物。
6. 问：适用于哪些细胞？
答：LumiDye™ Caspase 3 Substrate 已被报道用于多种原代细胞和科研文献中的永生化细胞中。
LumiDye™ 488 Caspase-3 Substrate provides an effective tool for detecting apoptosis based on caspase-3/7 activity, compatible with fluorescence microscopy and flow cytometry. Unlike other caspase fluorescent substrates or fluorescent inhibitors used in FLICA-based assays, LumiDye™ 488 Caspase-3 Substrate detects caspase-3/7 activity without inhibiting the apoptotic process in intact cells.
The substrate consists of a fluorescent DNA dye conjugated to the DEVD recognition sequence of caspase-3/7. The substrate is initially non-fluorescent and crosses the cell membrane into the cytoplasm. In apoptotic cells, caspase-3/7 cleaves the substrate to release a high-affinity DNA stain. This dye translocates to the nucleus, labels DNA, and emits bright green fluorescence. Therefore, LumiDye™ 488 Caspase-3 Substrate is bifunctional: it can detect caspase-3/7 activity and visualize morphological changes of the nucleus during apoptosis. LumiDye™ 488 staining is compatible with formaldehyde fixation and subsequent immunostaining experiments.
LumiDye™ 488 Caspase-3 Substrate is available in two formulations: DMSO and PBS. The PBS formulation is suitable for cells sensitive to DMSO toxicity. For cell types insensitive to DMSO, the DMSO formulation can be added to enhance staining efficiency during incubation with LumiDye™ 488.
Product Features:
⁕ Excellent stability: high fluorescence brightness and strong photobleaching resistance
⁕ Low batch-to-batch variation: consistent performance across batches
Product Specifications:
Ex/Em: 500/530 nm (with DNA)
Materials to be Provided by User:
1. Consumables: 1.5 mL centrifuge tubes
2. Equipment: Flow cytometer
Operating Procedure:
1. Experimental Optimization
The protocol described below is designed for endpoint assay. LumiDye™ 488 Substrate can also be applied in long-term cell incubation studies. Cell density, substrate concentration and inhibitor concentration may require optimization. The optimal substrate concentration generally ranges from 1 to 10 μM. Cells may be incubated with substrate in culture medium, PBS or other selected buffers. For adherent cells, replacement with fresh medium containing substrate is recommended to avoid heterogeneous background. Washing or medium exchange after substrate incubation is optional.
2. Controls
The following controls are recommended:
(1) Negative control: cells without apoptosis induction.
(2) Positive control: cells with apoptosis induction.
(3) Inhibitor control: cells induced for apoptosis while (or 10–30 min in advance) incubated with Caspase-3/7 inhibitor, followed by addition of LumiDye™ 488 Caspase 3 Substrate.
Ac-DEVD-CHO Caspase 3 Inhibitor Control:
The Caspase-3/7 inhibitor Ac-DEVD-CHO (Cat. No. A305146) can be used to confirm that the fluorescent signal of LumiDye™ 488 is Caspase-3/7-dependent. For inhibitor control, the final inhibitor concentration should be at least 2-fold of the substrate concentration (e.g., 10 μM Ac-DEVD-CHO when 5 μM LumiDye™ 488 substrate is used). Pre-incubate cells with Ac-DEVD-CHO at room temperature for 15–30 min before adding substrate, and maintain the inhibitor in the incubation medium after substrate addition. Ac-DEVD-CHO is a reversible competitive inhibitor. For certain cell types, irreversible Caspase-3/7 inhibitors such as Z-DEVD-FMK may be required, or the inhibitor should be added prior to or during apoptosis induction.
3. Flow Cytometry
(1) Induce cell apoptosis using an appropriate method; untreated cell samples serve as controls.
(2) For adherent cells, digest cells with trypsin or other methods prior to performing the LumiDye™ 488 Caspase 3 assay.
(3) Resuspend cells to a density of 10⁶ cells/mL.
(4) Transfer 0.2 mL cell suspension into a flow cytometry tube.
(5) For control samples, treat cells with Ac-DEVD-CHO (see Section 2, Ac-DEVD-CHO Caspase 3 Inhibitor Control).
(6) Add 5 μL of 0.2 mM Substrate into 200 μL cell suspension and mix immediately to reach a final substrate concentration of 5 μM. The optimal substrate concentration varies among cell types and needs empirical optimization.
(7) Incubate cells at room temperature in the dark for 15–30 min.
(8) Add 300 μL medium or PBS, then analyze by flow cytometer. Use the channel for green fluorescence detection (Ex/Em: 485/515 nm).
4. Fluorescence Microscopy
(1) Induce cell apoptosis using an appropriate method; untreated cell samples serve as controls.
(2) For inhibitor control samples, treat cells with Ac-DEVD-CHO (see Section 2, Ac-DEVD-CHO Caspase 3 Inhibitor Control).
(3) Replace medium with fresh culture medium or PBS containing 5 μM Substrate (see Section 1, Experimental Optimization). For inhibitor control groups, incubate cells with inhibitor and substrate simultaneously.
(4) Incubate cells at room temperature for 30 min or longer.
(5) Cells can be observed directly in substrate-containing medium. For endpoint assay, wash cells with PBS and observe under fluorescence microscope with green fluorescence filter set (Ex/Em: 485/515 nm).
5. Fluorescence Microplate Reader
(1) Culture adherent cells in black 96-well plates. For suspension cells, adjust cell density to 10⁶ cells/mL and dispense 0.2 mL cell suspension per well.
(2) Induce cell apoptosis using an appropriate method; untreated cell samples serve as controls.
Note: Cells may be treated in tubes or flasks before transferring to 96-well assay plates.
(3) For inhibitor control samples, treat cells with Ac-DEVD-CHO (see Section 2, Ac-DEVD-CHO Caspase 3 Inhibitor Control).
(4) For suspension cells, add Substrate directly and mix well. For adherent cells, replace medium with fresh culture medium or PBS containing 5 μM Substrate (see Section 1, Experimental Optimization). For inhibitor control groups, incubate cells with inhibitor and substrate simultaneously.
(5) Cells can be measured directly in substrate-containing medium.
(6) Gently agitate to resuspend suspension cells. Set the fluorescence microplate reader at excitation 488 nm and emission 520 nm. Bottom reading mode is recommended for adherent cells. Variations in adherent cell density may lead to inaccurate readings.
Precautions:
1. Briefly centrifuge the product to collect all contents at the bottom of the tube before use, then proceed with subsequent experiments.
2. Cells may be co-stained with Hoechst 33342 at a final concentration of 1 μM to produce blue nuclear fluorescence (Ex/Em: 346/460 nm).
3. LumiDye™ 488 staining is compatible with formaldehyde fixation but incompatible with methanol fixation.
4. Formaldehyde-fixed LumiDye™ 488-stained cells can be treated with 0.1% Triton X-100 for subsequent staining, though fluorescence intensity may decrease after treatment.
5. All fluorescent dyes are susceptible to photobleaching. Minimize light exposure to slow down fluorescence quenching.
6. This product is for research use only. It shall not be used for clinical diagnosis or therapy, food or pharmaceutical applications. Do not store this product in residential premises.
7. Wear a lab coat and disposable gloves during operation for your safety and health.
FAQ:
1. Q: What is the stability of this product?
A: This reagent exhibits excellent stability. User feedback shows that the product retains good performance after storage at 37 °C for 4–5 days.
2. Q: When should the substrate be added to cells?
A: The substrate can be added either early or late during the experiment. It does not interfere with the apoptotic process and enables real-time monitoring of Caspase-3 activity.
3. Q: Can it be used for tissue staining?
A: We have not performed live tissue staining assays with this product. Literature references indicate its use in embryonic tissues and 3D cultured cells.
4. Q: Is it compatible with subsequent immunostaining?
A: Yes. Fixation with 2%–4% paraformaldehyde at room temperature for 10–15 min is recommended. Excessively long fixation will lead to signal reduction.
5. Q: What about its specificity?
A: Similar to other Caspase-3 substrates, LumiDye™ Caspase 3 Substrate relies on the DEVD consensus sequence recognized by Caspase-7. Other caspases may also cleave DEVD substrates due to overlapping recognition sequences.
6. Q: Which cell types are suitable?
A: LumiDye™ Caspase 3 Substrate has been reported for use in multiple primary cells and immortalized cell lines described in research literature.
规格
规格或纯度
BioReagent, 适用于荧光分析, 1 mM in PBS
稳定性与储存
Store at -20℃ long term (36 months). Store in the dark.
英文名称
LumiDye™ 488 Caspase 3 Substrate (1 mM)
储存条件
避光,-20°C储存
运输条件
超低温运输
Excitation(nm)
Blue Laser(488nm)
Emission(nm)
530nm
Ex/Em(nm)
Ex:350nm;Em:461nm
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