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细胞凋亡诱导剂套装 (即用型) (U1524531) 包含五种经典细胞凋亡诱导剂,用于在哺乳动物细胞中诱导或调节细胞凋亡(程序性细胞死亡)。该套装以DMSO溶液形式提供,不含防腐剂。
1. 放线菌素D (Actinomycin D)
核心机制:抗肿瘤抗生素,通过插入DNA双链抑制RNA合成(转录抑制剂)。
典型应用场景:
mRNA稳定性分析(转录阻滞实验):加入放线菌素D终止新RNA合成后,检测特定mRNA随时间衰减情况,计算其半衰期。
转录调控研究:研究特定基因表达是否依赖于持续转录活性。
细胞凋亡模型建立:作为转录依赖性凋亡的诱导剂。
使用方法:
按1:1000稀释至培养基中(如1 μL储存液加入1 mL培养基),初始工作浓度为10 μM。
处理时间通常为6-24小时,具体需根据细胞类型优化。
参考浓度:文献报道放线菌素D诱导Vero细胞凋亡的浓度为0.25-4.0 mg/L,处理12-60小时。
2. 喜树碱 (Camptothecin)
核心机制:DNA拓扑异构酶I抑制剂,通过抑制Topo I活性导致DNA损伤,激活p53并上调TRAIL-R2(DR5)和Bak表达。
典型应用场景:
凋亡实验阳性对照:作为诱导细胞凋亡的通用方法,为各类细胞死亡检测实验(如Annexin V染色、TUNEL染色、Caspase活性检测等)提供可靠的阳性对照。
DNA损伤与修复研究:作为特异性Topo I抑制剂,用于研究DNA损伤响应及修复机制。
细胞周期与凋亡研究:喜树碱主要作用于S期细胞,常用于研究细胞周期检查点激活和凋亡通路。
抗癌药物机制研究:喜树碱及其衍生物(伊立替康、拓扑替康)是临床抗癌药物,用于抗癌机制或药物联用研究。
使用方法:
按1:1000稀释至培养基中,初始工作浓度为2 μM。
建议进行时间梯度实验以确定最佳处理时间,推荐 2-12 小时 孵育(37°C)。
参考浓度:文献报道50 nmol/L喜树碱处理U937细胞24小时即可诱导明显凋亡;100 nmol/L~10 μmol/L处理HeLa细胞24小时可见促凋亡效果。
3. 环己酰亚胺 (Cycloheximide)
核心机制:蛋白质合成抑制剂,通过抑制核糖体翻译过程阻断新蛋白合成。
典型应用场景:
蛋白质降解动力学(CHX Chase实验) :这是环己酰亚胺最独特和最重要的应用。加入环己酰亚胺终止新蛋白合成后,追踪特定已有蛋白水平随时间下降的速度,直接测定该蛋白的降解速率和半衰期。
信号通路研究:判断某个快速细胞反应是否需要新蛋白合成。
使用方法:
按1:1000稀释至培养基中,初始工作浓度为100 μM。
在CHX Chase实验中,通常在加入环己酰亚胺后0、2、4、6、8小时等时间点收集细胞裂解液,通过免疫印迹(Western Blot)检测目标蛋白剩余量。
参考浓度:文献中常用浓度为50-200 μg/mL。
4. 地塞米松 (Dexamethasone)
核心机制:合成糖皮质激素,通过结合并激活细胞内糖皮质激素受体诱导凋亡。
典型应用场景:
胸腺细胞及淋巴细胞凋亡研究:地塞米松是胸腺细胞凋亡的经典诱导剂。
免疫抑制与炎症研究:作为强效免疫抑制剂和抗炎药,广泛用于研究炎症信号通路(如NF-κB)和免疫细胞功能调控。
疾病模型构建:常用于构建糖皮质激素性骨质疏松等疾病模型。
使用方法:
按1:1000稀释至培养基中,初始工作浓度为10 μM。
处理时间通常为12-48小时。
参考浓度:文献报道0.5-2.0 μg/mL地塞米松处理SMMC-7721细胞24小时可诱导凋亡;0.1-10 μmol/L处理CEM细胞48小时可抑制增殖。
5. 依托泊苷 (Etoposide)
核心机制:鬼臼毒素衍生物,拓扑异构酶II抑制剂,可增强dsDNA和ssDNA裂解并抑制DNA再连接。
典型应用场景:
DNA损伤与修复研究:作为Topo II抑制剂造成DNA双链断裂,是研究DNA损伤修复(同源重组修复、非同源末端连接)的常用工具。
细胞周期与凋亡研究:在S期抑制细胞周期,在正常细胞系和肿瘤细胞系中诱导凋亡。
抗癌药物机制研究:依托泊苷是临床广泛使用的化疗药物,可用于研究其抗癌机制、耐药性或药物联用效果。
使用方法:
按1:1000稀释至培养基中,初始工作浓度为10 μM。
处理时间通常为16-48小时。
参考浓度:文献报道1.5-15 μM依托泊苷处理MEF细胞18小时即可诱导凋亡;100-500 μM处理多种肿瘤细胞24小时可显著诱导凋亡
总结:如何选择用哪种诱导剂?
本试剂盒中的五种诱导剂通过不同机制诱导凋亡,分别适用于不同的研究场景。请务必根据您的实验目的选择合适的诱导剂,切勿随意混用或替换。
研究转录或mRNA稳定性 → 放线菌素D
研究DNA损伤修复或作为凋亡阳性对照 → 喜树碱或依托泊苷
研究蛋白质降解动力学 → 环己酰亚胺(CHX Chase实验)
研究免疫抑制或糖皮质激素信号 → 地塞米松
重要提示:五种诱导剂的作用靶点和机制各不相同。放线菌素D和环己酰亚胺分别作用于转录和翻译层面,喜树碱与依托泊苷均靶向拓扑异构酶但亚型不同,地塞米松则通过核受体途径发挥作用。若不明确实验目的而随意选用,可能无法获得预期凋亡效果,甚至产生误导性结果。如果确实需要联合使用多种诱导剂,请务必先进行预实验,优化各组分浓度和处理时间,并设置完善的单药对照组。
产品组分
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使用方法
1、推荐起始浓度:建议使用所提供贮备液的1000倍稀释液作为诱导许多细胞培养系统中凋亡活性的起点(例如,在1 mL培养基中加入1 μL储备液)。最佳剂量可能因不同的细胞和培养条件而异。
2、小体积添加技巧:当需要将抑制剂添加到小于1 mL的体系中时,为避免加样误差,可以先将储备液用新鲜的DMSO进行1:10稀释,然后再用培养基或PBS进行1:100的稀释。
3、剂量优化:最佳诱导剂量会因细胞类型和培养条件的不同而异,建议根据实验体系进行优化。
注意事项
1、该试剂盒含有高毒性试剂,仅限专业用户使用。
2、在操作浓缩溶液和稀释溶液时,请佩戴手套和其他防护设备以及遵循良好的实验室规范。
3、应在-20°C下储存试剂盒物料直至其有效期,避免反复冻融循环。
4、该产品仅供研究使用,不得用于诊断或治疗目的。
The Apoptosis Inducer Set (Ready-to-use) (U1524531) contains five classic apoptosis inducers for inducing or regulating apoptosis (programmed cell death) in mammalian cells. The set is provided as a liquid in DMSO without preservatives
1. Actinomycin D
Mechanism: Antitumor antibiotic that intercalates into DNA and inhibits RNA synthesis (transcription inhibitor).
Typical Applications:
mRNA stability assay (transcriptional arrest): After adding Actinomycin D to block new RNA synthesis, measure the decay of a specific mRNA over time to calculate its half‑life.
Transcriptional regulation studies: Investigate whether gene expression depends on ongoing transcription.
Apoptosis model: Induce transcription‑dependent apoptosis.
Usage:
Dilute 1:1000 into culture medium (e.g., 1 μL stock into 1 mL medium). Initial working concentration = 10 μM.
Treatment time: typically 6-24 hours, but optimize according to cell type.
Reference range in literature: 0.25-4.0 mg/L for Vero cells, 12-60 hours.
2. Camptothecin
Mechanism: DNA topoisomerase I inhibitor. By inhibiting Topo I activity, it causes DNA damage, activates p53, and upregulates TRAIL‑R2 (DR5) and Bak expression.
Typical Applications:
Positive control for apoptosis assays: As a general method for inducing apoptosis, it provides a reliable positive control for various cell death detection assays (e.g., Annexin V staining, TUNEL staining, Caspase activity assays).
DNA damage and repair studies: As a specific Topo I inhibitor, it is used to study DNA damage response and repair mechanisms.
Cell cycle and apoptosis studies: Camptothecin primarily acts on S‑phase cells and is commonly used to investigate cell cycle checkpoint activation and apoptotic pathways.
Anticancer drug mechanism studies: Camptothecin and its derivatives (irinotecan, topotecan) are clinical anticancer agents; useful for studying anticancer mechanisms or drug combination effects.
Usage:
Dilute 1:1000 into culture medium. Initial working concentration = 2 μM.
Treatment time: typically 12-48 hours.
Reference range in literature: 50 nmol/L induces apoptosis in U937 cells at 24 h; 100 nmol/L-10 μmol/L effective in HeLa cells at 24 h.
3. Cycloheximide
Mechanism: Protein synthesis inhibitor that blocks translation by acting on ribosomes.
Typical Applications:
Protein degradation kinetics (CHX chase assay): This is its most distinctive and important use. After blocking new protein synthesis, track the decline of a pre‑existing protein over time to measure its degradation rate and half‑life.
Signal transduction studies: Determine whether a rapid cellular response requires de novo protein synthesis.
Usage:
Dilute 1:1000 into culture medium. Initial working concentration = 100 μM.
For CHX chase: collect cell lysates at multiple time points (e.g., 0, 2, 4, 6, 8 h) after addition and analyse by Western blot.
Reference range in literature: Commonly 50–200 μg/mL.
4. Dexamethasone
Mechanism: Synthetic glucocorticoid that binds and activates the intracellular glucocorticoid receptor to induce apoptosis.
Typical Applications:
Thymocyte and lymphocyte apoptosis: Classic inducer of apoptosis in thymocytes.
Immunosuppression and inflammation research: Potent anti‑inflammatory and immunosuppressive agent; widely used to study NF‑κB and immune cell regulation.
Disease modelling: Often used to establish glucocorticoid‑induced osteoporosis models, among others.
Usage:
Dilute 1:1000 into culture medium. Initial working concentration = 10 μM.
Treatment time: typically 12-48 hours.
Reference range in literature: 0.5-2.0 μg/mL in SMMC‑7721 cells at 24 h; 0.1-10 μmol/L in CEM cells at 48 h for proliferation inhibition.
5. Etoposide
Mechanism: Podophyllotoxin derivative; topoisomerase II inhibitor that enhances dsDNA and ssDNA cleavage and prevents DNA religation.
Typical Applications:
DNA damage and repair studies: Causes DNA double‑strand breaks; a common tool to study homologous recombination and non‑homologous end joining repair.
Cell cycle and apoptosis: Arrests cells in S phase and induces apoptosis in both normal and tumour cell lines.
Anticancer drug mechanism: Clinically used chemotherapeutic; useful for studying its anticancer action, resistance, or combination therapies.
Usage:
Dilute 1:1000 into culture medium. Initial working concentration = 10 μM.
Treatment time: typically 16-48 hours.
Reference range in literature: 1.5-15 μM in MEF cells at 18 h; 100-500 μM in various tumour cells at 24 h for significant apoptosis.
Summary: How to Choose Which Inducer to Use?
The five inducers in this kit induce apoptosis through distinct mechanisms and are suitable for different research scenarios. Please select the appropriate inducer according to your experimental objectives – do not mix or substitute them arbitrarily.
Transcription or mRNA stability studies → Actinomycin D
DNA damage and repair studies, or as a positive control for apoptosis → Camptothecin or Etoposide
Protein degradation kinetics studies → Cycloheximide (CHX chase assay)
Immunosuppression or glucocorticoid signaling studies → Dexamethasone
Important Note: These five inducers have completely different targets and mechanisms. Actinomycin D and Cycloheximide act at the transcriptional and translational levels, respectively; Camptothecin and Etoposide both target topoisomerases but with different subtypes; Dexamethasone functions through the nuclear receptor pathway. Using an incorrect inducer without a clear purpose may fail to achieve the expected apoptotic effect or may lead to misleading results. If combination treatment with multiple inducers is required, please perform pilot experiments to optimize concentrations and treatment times, and include appropriate single‑agent controls.
Components
WARNING: This kit contains highly toxic reagents. For professional users only. Wear gloves and other protective equipment and follow good laboratory practices when handling concentrated and diluted solutions. |
| U1524531 | 组件 | 物理外观 | 1 kit | 储存 | 单次测试使用量 |
| U1524531A | 放线菌素D (10 mM) | 液体 | 50 μL | -20℃,避光 | 建议1/1000稀释 |
| U1524531B | 喜树碱 (2 mM) | 液体 | 1 mL | -20℃,避光 | 建议1/1000稀释 |
| U1524531C | 环己酰亚胺 (100 mM) | 液体 | 1 mL | -20℃,避光 | 建议1/1000稀释 |
| U1524531D | 地塞米松 (10 mM) | 液体 | 1 mL | -20℃,避光 | 建议1/1000稀释 |
| U1524531E | 依托泊苷 (10 mM) | 液体 | 100 μL | -20℃,避光 | 建议1/1000稀释 |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
| 分析证书 | U1524531 |
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