活细胞Caspase-3/7活性与Annexin V双重凋亡检测试剂盒通过整合LumiDye™ 488 Caspase-3/7底物、LumiDye™ 647标记的Annexin V、Hoechst 33342，构建了集约简便的活细胞凋亡多参数同步检测方案。其核心机制在于：
(1) LumiDye™ 488 Caspase-3/7底物作为非荧光膜渗透性底物，被凋亡细胞内激活的Caspase-3/7特异性剪切后，释放高亲和性DNA染料并发出绿色荧光，同步实现酶活性监测与核形态变化分析；
(2) LumiDye™ 647 Annexin V则通过结合外翻的磷脂酰丝氨酸 (PS)将凋亡细胞标记为红色；
(3) 借助Hoechst 33342蓝色染料的全细胞穿透性，对所有细胞的细胞核进行普遍性染色，作为细胞计数与定量校正的稳定内参。
相较于传统传统检测方法如DEVD-R110荧光底物需裂解细胞，无法用于活细胞检测且仅反映群体平均活性，FLICA试剂作为不可逆抑制剂会干扰酶功能并限制实时监测。本试剂盒优势显著：通过活细胞一步染色设计，结合荧光亮度提升50%的优化方案，显著增强检测灵敏度，无需裂解细胞，可对单细胞进行动态追踪；一次染色同步区分健康细胞、凋亡早期细胞、凋亡晚期细胞及坏死细胞，实现“四态”同步鉴别；兼容多通道检测；操作流程简化，从染色到检测仅需15分钟。本试剂盒适用于流式细胞术、荧光显微镜或其它荧光检测系统进行检测，为研究提供了灵活选择，突破了传统终点检测的局限。能在单细胞水平同步量化Caspase-3/7活性、PS外翻、坏死及健康状态，精准解析凋亡异质性，尤其可识别Caspase-3/7高活性但PS未外翻的早期凋亡亚群。
细胞凋亡的调控失衡与神经退行性疾病 (如阿尔茨海默病中神经元的过度凋亡)、心肌梗死 (缺血导致心肌细胞凋亡)等病理过程密切相关。该试剂盒的核心价值在于揭示肿瘤或药物处理后细胞群体的差异化响应机制，为药物开发 (如评估化合物凋亡动力学及毒性)和疾病机制研究 (如肿瘤异质性、神经退行性疾病模型)提供精准工具。通过单细胞分辨率同步解析Caspase-3/7活性爆发、PS外翻时序及坏死事件，不仅深化了对凋亡分子网络的理解，也为靶向治疗策略的制定提供了关键数据支持。
注：效能等同Biotium NucView® 488 and CF®640R Annexin V Dual Apoptosis Assay Kit for Live Cells (#30037)；NucView®、CF® 640R均是Biotium的商标及注册商标。
应用范围：贴壁细胞或悬浮细胞中细胞凋亡、坏死及健康多维度状态监测；活细胞动态追踪凋亡进程、区分死亡模式；药物开发、疾病机制研究；识别凋亡亚群 (如Caspase-3/7高活性但PS未外翻的早期凋亡细胞)，解析肿瘤/药物处理后的细胞异质性响应。
产品特点：
1. 活细胞免裂解检测：活细胞适用，无需细胞裂解步骤；
2. 单细胞多维度分析：提供单个细胞的Caspase-3/7活性、PS外翻、坏死及健康状态；
3. 实时动态追踪：实时捕捉Caspase-3/7活性全程，可逆底物不抑制酶活；
4. 快速可视化核形态：15 min一步染色，直观地观察到凋亡细胞核的形态变化。
产品参数：
1. LumiDye™ 488 Caspase-3/7底物 Ex/Em：500/530 nm
2. LumiDye™ 647-Annexin V Ex/Em：650/665 nm
3. Hoechst 33342 Ex/Em：350/461 nm (with DNA)
产品组分：
| L1520213 | 组件 | 10 T | 50 T | 100 T | Storage | | L1520213A | LumiDye™ 488 Caspase-3/7底物 | 50 μL | 250 μL | 500 μL | 2-8℃. Store in the dark. | | L1520213B | LumiDye™ 647-Annexin V | 50 μL | 250 μL | 500 μL | 2-8℃. Store in the dark. | | L1520213C | 1× Annexin V结合缓冲液 | 8 mL | 2×50 mL | 3×50 mL | 2-8℃. Store in the dark. | | L1520213D | Caspase-3/7抑制剂 | 10 μL | 50 μL | 100 μL | 2-8℃. Store in the dark. | | L1520213E | Hoechst 33342 | 10 μL | 50 μL | 100 μL | 2-8℃. Store in the dark. |
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注：100T为试剂盒使用次数为进行96孔板荧光显微镜检测时可检测的微孔总数。
注意事项：
1. 使用贴壁细胞进行实验时，使用胰酶进行消化后的细胞，必须使用含有血清的培养基中和并去除胰酶，否则胰酶将会降解Annexin V，导致染色失败。
2. 本实验中不建议使用甲醛固定细胞，Annexin V依赖于正常细胞中的完整细胞膜进行染色。若需固定需添加氯化钙。
3. 建议在实验中设置单染色的对照组，流式检测中用于调节补偿。
4. 本产品仅限于科研用途并且不得存放于普通住宅内。
5. 为了您的安全和健康，请遵循您所在常规实验室安全规定。
使用说明：
一、 实验前准备
1. 试剂准备：
将试剂盒各组分恢复至室温，准备足量PBS缓冲液。
2. 仪器准备：
(1) 荧光显微镜：激发/发射波长：Ex/Em：500/530 nm、Ex/Em：650/665 nm、Ex/Em：350/461 nm；
(2) 流式细胞仪：激发/发射波长：Ex/Em：500/530 nm、Ex/Em：650/665 nm、Ex/Em：350/461 nm。
3. 对照组设置：
| 组别 | LumiDye™ 488 Caspase-3/7底物 | LumiDye™ 647-Annexin V | Hoechst 33342 | Caspase-3/7抑制剂 | 样品细胞 | | 阴性对照 | + | + | + | - | 不做任何处理 | | 单阳对照 | + | - | - | - | 明显凋亡 | | 单阳对照 | - | + | - | - | 明显凋亡 | | 单阳对照 | - | - | + | - | 明显凋亡 | | 单阳对照 | - | - | - | - | 明显凋亡 | | 抑制对照 | + | + | + | + | 明显凋亡 | | 实验组 | + | + | + | - | 实验组细胞 |
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(1) 阴性对照组：排除染料的非特异性结合，验证染色的特异性。
(2) 单阳对照组：排除荧光串扰，校准信号强度参数、流式检测中调节补偿。
(3) 抑制对照组：验证LumiDye™ 488荧光信号对Caspase-3/7的依赖性。
二、 操作步骤
方案一：荧光显微镜检测 (适用于悬浮细胞)
1. 细胞处理：
(1) 按照所需的实验设计诱导细胞凋亡。
(2) 抑制对照处理：凋亡诱导前10-15 min加入1 μL组分D (Caspase-3/7抑制剂)，再加凋亡诱导剂。
注：抑制剂按照悬浮细胞数量约1.5×10⁵个细胞使用量为1 μL的比例按实际细胞数量进行添加。
2. 收集与洗涤细胞：
(1) 收集待测悬浮细胞悬液至离心管中。
(2) 1000 rpm，室温离心5 min，小心吸弃上清液。
3. 细胞重悬与计数：
(1) 使用PBS重悬并细胞计数。
(2) 取5×10⁴-10⁵个细胞，1000 rpm低速离心5 min，去除PBS。
(3) 使用100 μL组分C (1× Annexin V结合缓冲液)重悬。1000 rpm低速离心5 min，去除上清。
注：显微镜法对细胞密度要求不严格，只要实验组与对照组密度一致即可。具体可根据样本种类与实验条件灵活调整，以显微镜下视野中细胞分布清晰可辨为宜。
4. 细胞染色：
(1) 取100 μL组分C重悬细胞，加入5 μL组分A (LumiDye™ 488 Caspase-3/7底物)，加入5 μL组分B (LumiDye™647 AnnexinV)，加入1 μL组分E (Hoechst 33342)，轻轻吹打混匀。
(2) 室温避光孵育15-30 min (孵育时间可根据预实验结果优化)。
注：各染料的使用量可以根据不同的细胞种类、预实验或实验条件设置的使用量进行调整，但组分A (LumiDye™ 488 Caspase-3/7底物)和组分D (Caspase-3/7抑制剂)的使用量保持体积比5: 1。
5. 洗涤与重悬：
(1) 孵育结束后，1000 rpm，室温离心5 min，收集细胞。
(2) 小心吸弃含染料的上清液 (此为废液，需妥善处理)。
(3) 加入100 μL组分C (1× Annexin V结合缓冲液)轻柔重悬细胞，进行洗涤，吸净上清液。
(4) 加入100 μL组分C (1× Annexin V结合缓冲液)轻柔重悬细胞，制成待测样品。
注：若不及时检测，可将离心管暂时放置于冰上。
6. 显微镜观察与拍照：
(1) 96孔板法：将100 μL细胞悬液加入96孔板中，静置片刻，待细胞自然沉降贴底后，置于荧光显微镜下观察。
注：细胞悬液使用量可以根据细胞计数的数量自行调整，显微镜视野中细胞分布70%-80%为最佳。
(2) 载玻片法：取20-30 μL细胞悬液滴加于洁净载玻片上，轻轻盖上盖玻片，避免产生气泡，置于荧光显微镜下观察。
(3) 分别在预设的滤光片条件下，寻找并分别拍摄。
LumiDye™ 488 Caspase-3/7底物检测信号：选用 FITC 通道 (Ex/Em：490/525 nm)或相应滤光片；
LumiDye™ 647 Annexin V检测信号：选用APC通道 (Ex/Em：650/665 nm)或相应滤光片；
Hoechst 33342检测信号：选用DAPI通道 (Ex/Em：350/461 nm)或相应滤光片。
方案二：荧光显微镜检测 (适用于贴壁细胞)
1. 抑制对照处理：
(1) 提前一天在孔板中接种细胞，使得细胞汇合度达到70%-85%，待细胞贴壁后按照实验设计处理细胞。
(2) 凋亡诱导前10-15 min，加入1 μL组分D (Caspase-3/7抑制剂)，再加凋亡诱导剂。
注：抑制剂按照悬浮细胞数量约1.5×10⁵个细胞使用量为1 μL的比例按实际细胞数量进行添加。
2. 细胞清洗：
(1) 直接在培养板中吸弃细胞培养液，PBS清洗一次。
(2) 根据培养板规格，加入对应体积的组分C (1× Annexin V结合缓冲液)轻柔洗涤细胞一次 (例如：96孔板加100 μL/孔，48孔板加150 μL/孔，24孔板加250 μL/孔，12孔板加500 μL/孔，6孔板加1 mL/孔)。洗涤后吸净组分C。
3. 制备染色工作液：
按照每100 μL中分别添加组分A (LumiDye™ 488 Caspase-3/7底物) 5 μL、组分B (LumiDye™ 647-Annexin V) 5 μL、组分E (Hoechst 33342) 1 μL的使用量将染料加入到组分C (1× Annexin V结合缓冲液)中，混匀备用。
注：各染料的使用量可以根据不同的细胞种类、预实验或实验条件设置的使用量进行调整，但组分A和组分D的使用量保持体积比5: 1。
4. 细胞染色：
(1) 向各孔中加入对应体积的染色工作液 (体积同步骤1，例如：96孔板加100 μL/孔，48孔板加150 μL/孔，24孔板加250μL/孔，12孔板加500 μL/孔，6孔板加1 mL/孔)。
(2) 室温避光孵育15-30 min (孵育时间可根据预实验结果优化）。
5. 洗涤：
(1) 孵育结束后，吸弃各孔中的染色工作液。
(2) 按步骤2的体积，加入组分C (1× Annexin V结合缓冲液)洗涤细胞。
6. 显微镜观察与拍照：
(1) 洗涤后，每孔加入对应体积的组分C (1× Annexin V结合缓冲液)，以保持细胞湿润。
(2) 直接将培养板置于荧光显微镜下观察和拍照。滤光片设置同方案一。
方案三：流式细胞仪检测 (适用于悬浮与贴壁细胞)
1. 收集与制备单细胞悬液：
(1) 悬浮细胞：同悬浮细胞：同方案一，最终取5×10⁵-10⁶ 个细胞。
(2) 贴壁细胞：
a. 将细胞培养基吸出至适合的离心管内备用。贴壁细胞用少量PBS清洗一遍，清洗后的PBS吸出后同样转移到盛装培养基的离心管内，1000 rpm，室温离心5 min。
b. 加入适量不含EDTA的胰酶消化液消化细胞。
注：胰酶使用量、消化时间、消化温度根据各自实验室的经验操作。
c. 关键：加入a步骤离心管内备用的细胞培养基，终止消化。
d. 将细胞悬液转移至合适的离心管，1000 rpm，室温离心5 min，吸弃上清。
e. 收集a步骤与d步骤中的细胞；
f. 用PBS重悬细胞并进行细胞计数；
g. 取5×10⁴-10⁵个细胞，1000 rpm，室温离心5 min，吸弃上清；
h. 100 μL 组分C (1× Annexin V结合缓冲液)清洗细胞，1000 rpm，室温离心5 min，吸弃上清。
注：胰酶消化步骤对于贴壁细胞很关键。消化时间过短，细胞需要用力吹打才能脱落容易造成机械损伤，导致细胞凋亡的假阳性；消化时间过长，同样会造成细胞凋亡的假阳性，影响细胞膜上PS与Annexin V的结合干扰细胞凋亡的检测。
2. 细胞染色：
(1) 用100 μL组分C (1× Annexin V结合缓冲液)重悬细胞。
(2) 加入5 μL组分A (LumiDye™ 488 Caspase-3/7底物)，加入5 μL组分B (LumiDye™ 647-Annexin V)，加入1 μL组分E(Hoechst 33342)，轻轻吹打混匀。
(3) 室温避光孵育15-30 min (孵育时间可根据预实验结果优化)。
注：各染料的使用量可以根据不同的细胞种类、预实验或实验条件设置的使用量进行调整，但组分A (LumiDye™ 488 Caspase-3/7底物)和组分D (Caspase-3/7抑制剂)的使用量保持体积比5: 1。
3. 洗涤与重悬：
(1) 孵育结束后，1000 rpm，室温离心5 min，收集细胞。
(2) 吸弃上清液，加入100 μL组分C (1× Annexin V结合缓冲液)轻柔重悬，进行洗涤。
(3) 再次离心，彻底吸弃上清液。
(4) 加入500 μL组分C (1× Annexin V结合缓冲液)轻柔重悬细胞，制成流式上机样品。
注：将待测样品置于冰上。
4. 上机检测：
(1) 重要：染色完成后1 h内进行流式检测，以保证荧光信号稳定。
(2) 用流式细胞仪在预设的检测通道下进行分析。
(3) 首先通过空白对照 (未染色细胞)调节电压，使细胞群位于坐标系左下角。
(4) 然后检测阴性对照和阳性对照，确认实验体系成立。
(5) 最后检测实验样品，记录并分析细胞的平均荧光强度。
LumiDye™ 488 Caspase-3/7底物 检测信号：选用FITC通道 (Ex/Em：490/525 nm)；
LumiDye™ 647-Annexin V 检测信号：选用APC通道 (Ex/Em：650/665 nm)；
Hoechst 33342 检测信号：选用DAPI通道 (Ex/Em：350/461 nm)。
三、 结果判读：
定性分析 (显微镜)：
图1. 活细胞Caspase-3/7活性与Annexin V双重凋亡检测试剂盒荧光镜检拍照
(1) 阴性组：K562正常细胞，进行LumiDye™ 488 Caspase-3/7底物、LumiDye™ 647-Annexin V、Hoechst 33342同时共染后合成画面Merge；
(2) 阳性组：诱导凋亡处理后的K562细胞，进行LumiDye™ 488 Caspase-3/7底物、LumiDye™ 647-Annexin V、Hoechst 33342同时共染后合成画面Merge；
(3) 抑制组：诱导凋亡并抑制Caspase-3/7活性处理后的K652细胞，进行LumiDye™ 488 Caspase-3/7底物、LumiDye™ 647-Annexin V、Hoechst 33342同时共染后合成画面Merge。
结果判读：在经过凋亡诱导处理后，与阴性对照相比Caspase-3/7活性明显增加 (绿色荧光亮度、细胞个数显著增强)，磷脂酰丝氨酸 (PS)外翻水平显著增加 (红色荧光亮度、细胞个数显著增强)，表明Caspase-3/7活性被激活、PS外翻显著、凋亡细胞数量增加。在加入Caspase-3/7抑制剂后，与阳性组对比Caspase-3/7活性明显降低 (绿色荧光亮度、细胞个数显著降低)，PS外翻水平显著降低，但与阴性组对比，抑制组高于阴性组的PS外翻水平及凋亡细胞数，结果表明在抑制Caspase-3/7活性后，会激活其他的凋亡通路。
注：实际检测结果可能因检测仪器、操作条件、细胞类型等差异产生不同，本示例仅提供参考。
常见疑问与解答：
1. 问：可以在染色前或染色后使用固定细胞吗？
答：要观察凋亡细胞的不同状态，只能用活细胞诱导凋亡后进行检测。如果将细胞进行固定，细胞在固定过程中将全部死亡，染色结果将出现大量的假阳性凋亡或信号。
2. 问：使用贴壁细胞进行流式检测时为什么要保留培养细胞的培养基？
答：药物处理后的细胞已经存在凋亡细胞，这些细胞的贴壁能力逐渐降低，会游离在培养基中，染色检测时需将这部分细胞考虑进去，否则检测结果中可能凋亡或坏死细胞的比例会比较低，对药物效果或者细胞处理效果的评价会产生错误判断；且细胞培养基中含有的血清能够高效的中和胰酶，残留的胰酶会降解后续染色过程中的Annexin V，导致染色失败。
3. 问：为什么Caspase-3/7抑制剂在细胞中没有抑制LumiDye™ 488染色？
答：Caspase-3/7抑制剂是一种可逆的竞争性抑制剂，细胞渗透性有限，可能不足以阻断非常高的Caspase-3/7活性水平。根据我们的经验，抑制剂处理可能会降低LumiDye™染色的细胞荧光强度，但通常不会降低到未经处理的对照细胞的水平。在凋亡诱导之前或之后添加像Z-DEVD-FMK这样的不可逆抑制剂可能更有效地抑制Caspase活性。
4. 问：在进行双重凋亡分析后，能固定细胞吗？
答：可以。建议使用2-4%的对甲醛在室温下固定10-15 min。过度固定可能导致信号减少。为了保留Annexin V染色，应在所有缓冲液中加入1.25 mM CaCl₂。不建议使用甲醇固定。LumiDye™ 488 染色可以承受0.1%Triton X-100的通透化处理，以便进行后续的免疫染色，信号强度在通透化和洗涤后可能会降低。Annexin V染色不能承受去污剂通透化。
6. 问：LumiDye™ Caspase-3/7底物对Caspase-3/7的特异性如何？
答：与其他Caspase-3/7底物一样，LumiDye™ Caspase-3/7底物基于DEVD Caspase-3/7共识序列，该序列也可以被Caspase-7切割。由于Caspases之间底物特异性的重叠，其他Caspases也可能切割DEVD底物。
7. 问：可以使用哪些细胞类型与LumiDye™ 488 Caspase-3/7底物一起使用？
答：LumiDye™ 488 Caspase-3/7底物在已发表的科学文献中被报道在多种原代细胞和永生化细胞系中有效。
8. 问：双重凋亡分析能用于组织染色吗？
答：双重凋亡分析不能用于固定细胞或组织切片。我们尚未验证该分析用于活组织染色。
Live Cell Caspase-3/7 Activity and Annexin V Dual Apoptosis Detection Kit integrates LumiDye™ 488 Caspase-3/7 Substrate, LumiDye™ 647-labeled Annexin V, and Hoechst 33342 to establish a compact and convenient solution for simultaneous multi-parameter detection of apoptosis in live cells. Its core mechanisms are as follows:
(1) As a non-fluorescent, membrane-permeable substrate, LumiDye™ 488 Caspase-3/7 Substrate is specifically cleaved by activated Caspase-3/7 in apoptotic cells, releasing a high-affinity DNA dye that emits green fluorescence, enabling simultaneous monitoring of enzymatic activity and analysis of nuclear morphological changes;
(2) LumiDye™ 647 Annexin V labels apoptotic cells red by binding to phosphatidylserine (PS) exposed on the cell surface;
(3) With the full cell permeability of the blue dye Hoechst 33342, it universally stains the nuclei of all cells, serving as a stable internal reference for cell counting and quantitative calibration.
Compared with traditional detection methods-such as the DEVD-R110 fluorescent substrate, which requires cell lysis, is incompatible with live-cell assays, and only reflects the average activity of cell populations; and FLICA reagents, which act as irreversible inhibitors that interfere with enzyme function and restrict real-time monitoring-this kit offers distinct advantages. Featuring a one-step live-cell staining protocol combined with an optimized formulation that increases fluorescence brightness by 50%, it significantly improves detection sensitivity without cell lysis and allows dynamic tracking at the single-cell level. A single staining step simultaneously distinguishes healthy, early apoptotic, late apoptotic, and necrotic cells, achieving simultaneous identification of four cellular states. It is compatible with multi-channel detection and has a simplified workflow, requiring only 15 minutes from staining to detection. Suitable for flow cytometry, fluorescence microscopy, or other fluorescence detection systems, this kit provides flexible options for research and overcomes the limitations of traditional endpoint assays. It enables simultaneous quantification of Caspase-3/7 activity, PS externalization, necrosis, and viability at the single-cell level, allowing precise analysis of apoptotic heterogeneity, especially the identification of early apoptotic subpopulations with high Caspase-3/7 activity but no PS externalization.
Dysregulated apoptosis is closely associated with pathological processes including neurodegenerative diseases (e.g., excessive neuronal apoptosis in Alzheimer’s disease) and myocardial infarction (cardiomyocyte apoptosis induced by ischemia). The core value of this kit lies in revealing the differential response mechanisms of cell populations following tumorigenesis or drug treatment, providing a precise tool for drug development (e.g., evaluating compound apoptotic kinetics and toxicity) and disease mechanism research (e.g., tumor heterogeneity and neurodegenerative disease models). By simultaneously resolving the timing of Caspase-3/7 activity bursts, PS externalization, and necrotic events at single-cell resolution, it not only deepens the understanding of the apoptotic molecular network but also provides critical data to support the development of targeted therapeutic strategies.
Note: Performance is equivalent to Biotium NucView® 488 and CF®640R Annexin V Dual Apoptosis Assay Kit for Live Cells (#30037). NucView® and CF® 640R are trademarks and registered trademarks of Biotium.
Applications
Multi-dimensional monitoring of apoptosis, necrosis, and viability in adherent or suspension cells; dynamic tracking of apoptotic progression in live cells and classification of cell death modes; drug development and disease mechanism research; identification of apoptotic subpopulations (e.g., early apoptotic cells with high Caspase-3/7 activity but no PS externalization); and analysis of heterogeneous cellular responses after tumorigenesis or drug treatment.
Product Features
1. Lysis-free live-cell detection: Suitable for live cells without cell lysis;
2. Single-cell multi-dimensional analysis: Simultaneously assesses Caspase-3/7 activity, PS externalization, necrosis, and viability in individual cells;
3. Real-time dynamic tracking: Captures the full course of Caspase-3/7 activity in real time; reversible substrate does not inhibit enzymatic activity;
5. Rapid visualization of nuclear morphology: One-step staining in 15 minutes allows direct observation of morphological changes in apoptotic nuclei.
Product Parameters
1. LumiDye™ 488 Caspase-3/7 Substrate Ex/Em: 500/530 nm
2. LumiDye™ 647-Annexin V Ex/Em: 650/665 nm
3. Hoechst 33342 Ex/Em: 350/461 nm (with DNA)
Product Components
| L1520213 | Component | 10 T | 50 T | 100 T | Storage | | L1520213A | LumiDye™ 488 Caspase-3/7 Substrate | 50 μL | 250 μL | 500 μL | 2-8℃. Store in the dark. | | L1520213B | LumiDye™ 647-Annexin V | 50 μL | 250 μL | 500 μL | 2-8℃. Store in the dark. | | L1520213C | 1× Annexin Binding Buffer | 8 mL | 2×50 mL | 3×50 mL | 2-8℃. Store in the dark. | | L1520213D | Caspase-3/7 Inhibitor | 10 μL | 50 μL | 100 μL | 2-8℃. Store in the dark. | | L1520213E | Hoechst 33342 | 10 μL | 50 μL | 100 μL | 2-8℃. Store in the dark. |
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Note: 100 T refers to the total number of wells that can be tested when using the kit for fluorescence microscopy assays in 96-well plates.
Precautions
1. When performing experiments with adherent cells, cells digested with trypsin must be neutralized and resuspended in serum-containing medium to remove trypsin. Otherwise, trypsin will degrade Annexin V and result in failed staining.
2. Fixing cells with formaldehyde is not recommended in this assay, as Annexin V staining relies on intact cell membranes. If fixation is required, calcium chloride (CaCl₂) must be added.
3. Single-stained control groups are recommended for compensation adjustment in flow cytometry.
4. This product is for research use only and must not be stored in ordinary residential premises.
5. For your safety and health, please comply with the general laboratory safety regulations in your facility.
Instructions for Use
I. Pre-Experiment Preparation
1. Reagent Preparation
1. Reagent PreparationAllow all kit components to equilibrate to room temperature, and prepare an adequate volume of PBS buffer.
2. Instrument Preparation
(1) Fluorescence microscope: excitation/emission wavelengths at Ex/Em: 500/530 nm, 650/665 nm, 350/461 nm, and 532/625 nm;
(2) Flow cytometer: excitation/emission wavelengths at Ex/Em: 500/530 nm, 650/665 nm, 350/461 nm, and 532/625 nm.
3. Control Group Setup
| Group | LumiDye™ 488 Caspase-3/7 Substrate | LumiDye™ 647-Annexin V | Hoechst 33342 | Caspase-3/7 Inhibitor | Sample Cells | | Negative Control | + | + | + | - | No treatment | | Single Positive Control | + | - | - | - | Apoptotic cells | | Single Positive Control | - | + | - | - | Apoptotic cells | | Single Positive Control | - | - | + | - | Apoptotic cells | | Single Positive Control | - | - | - | - | Apoptotic cells | | Inhibition Control | + | + | + | + | Apoptotic cells | | Experimental Group | + | + | + | - | Experimental cells |
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(1) Negative control group: To exclude non-specific binding of dyes and verify the specificity of staining.
(2) Single-positive control groups: To eliminate fluorescence crosstalk, calibrate signal intensity parameters, and adjust compensation in flow cytometry.
(3) Inhibition control group: To verify the dependence of LumiDye™ 488 fluorescence signal on Caspase-3/7 activity.
II. Procedures
Protocol A: Fluorescence Microscopy (for Suspension Cells)
1. Cell Treatment
(1) Induce cell apoptosis according to your experimental design.
(2) Inhibition control treatment: Add 1 μL of Component D (Caspase-3/7 Inhibitor) 10-15 min before apoptosis induction, followed by the apoptotic inducer.
Note: The inhibitor is added at a ratio of 1 μL per approximately 1.5×10⁵ suspension cells, adjusted according to actual cell number.
2. Cell Collection and Washing
(1) Collect the cell suspension to be tested into a centrifuge tube.
(2) Centrifuge at 1000 rpm for 5 min at room temperature, and carefully aspirate the supernatant.
3. Cell Resuspension and Counting
(1) Resuspend cells in PBS and perform cell counting.
(2) Take 5×10⁴-1×10⁵ cells, centrifuge at 1000 rpm for 5 min, and remove PBS.
(3) Resuspend in 100 μL of Component C (1× Annexin V Binding Buffer), centrifuge at 1000 rpm for 5 min, and discard the supernatant.
Note: Microscopy assays are not strict on cell density, as long as densities are consistent between experimental and control groups. The volume can be flexibly adjusted based on cell type and experimental conditions, to achieve clear and distinguishable cell distribution under microscopy.
4. Cell Staining
(1) Resuspend cells in 100 μL of Component C (1× Annexin V Binding Buffer). Add 5 μL of Component A (LumiDye™ 488 Caspase-3/7 Substrate), 5 μL of Component B (LumiDye™ 647 Annexin V), 1 μL of Component E (Hoechst 33342). Mix gently by pipetting.
(2) Incubate at room temperature for 15-30 min in the dark (incubation time can be optimized based on preliminary experiments).
Note: Dye volumes can be adjusted according to cell type, preliminary tests, or experimental conditions, but the volume ratio of Component A (LumiDye™ 488 Caspase-3/7 Substrate) to Component D (Caspase-3/7 Inhibitor) must remain 5:1.
5. Washing and Resuspension
(1) After incubation, centrifuge at 1000 rpm for 5 min at room temperature to collect cells.
(2) Carefully aspirate the dye-containing supernatant (waste liquid, to be disposed properly).
(3) Add 100 μL of Component C (1× Annexin V Binding Buffer), gently resuspend cells to wash, and completely aspirate the supernatant.
(4) Add 100 μL of Component C (1× Annexin V Binding Buffer) and gently resuspend cells to prepare the sample for detection.
Note: If not detected immediately, the centrifuge tube may be kept on ice temporarily.
6. Microscopic Observation and Imaging
(1) 96-well plate method: Add 100 μL of cell suspension to a 96-well plate, let stand briefly until cells settle naturally to the bottom, then observe under a fluorescence microscope.
Note: The volume of cell suspension can be adjusted based on cell count; optimal cell distribution is 70%-80% in the microscopy field of view.
(2) Slide method: Pipette 20-30 μL of cell suspension onto a clean glass slide, gently cover with a coverslip avoiding bubbles, and observe under a fluorescence microscope.
(3) Locate and capture images under preset filter conditions respectively:
LumiDye™ 488 Caspase-3/7 Substrate signal: FITC channel (Ex/Em: 490/525 nm) or equivalent filter;
LumiDye™ 647 Annexin V signal: APC channel (Ex/Em: 650/665 nm) or equivalent filter;
Hoechst 33342 signal: DAPI channel (Ex/Em: 350/461 nm) or equivalent filter;
Protocol B: Fluorescence Microscopy (for Adherent Cells)
1. Inhibition Control Treatment
(1) Seed cells in a plate one day in advance to reach 70%-85% confluence. After cells adhere, treat them according to the experimental design.
(2) Add 1 μL of Component D (Caspase-3/7 Inhibitor) 10-15 min before apoptosis induction, followed by the apoptotic inducer.Note: The inhibitor is added at a ratio of 1 μL per approximately 1.5×10⁵ suspension cells, adjusted according to actual cell number.
2. Cell Washing
(1) Aspirate the cell culture medium directly from the plate and wash once with PBS.
(2) Add an appropriate volume of Component C to gently wash the cells according to the plate type (e.g., 100 μL/well for 96-well plates, 150 μL/well for 48-well plates, 250 μL/well for 24-well plates, 500 μL/well for 12-well plates, 1 mL/well for 6-well plates). Aspirate Component C completely after washing.
3. Preparation of Staining Working Solution
Add dyes into Component C (1× Annexin V Binding Buffer) at the following volumes per 100 μL: 5 μL of Component A (LumiDye™ 488 Caspase-3/7 Substrate), 5 μL of Component B (LumiDye™ 647-Annexin V), 1 μL of Component E (Hoechst 33342). Mix well before use.
Note: Dye volumes can be adjusted according to cell type, preliminary tests, or experimental conditions, but the volume ratio of Component A (LumiDye™ 488 Caspase-3/7 Substrate) to Component D (Caspase-3/7 Inhibitor) must remain 5:1.
4. Cell Staining
(1) Add the corresponding volume of staining working solution to each well (same volume as in Step 1; e.g., 100 μL/well for 96-well plates, 150 μL/well for 48-well plates, 250 μL/well for 24-well plates, 500 μL/well for 12-well plates, 1 mL/well for 6-well plates).
(2) Incubate at room temperature for 15-30 min in the dark (incubation time can be optimized based on preliminary experiments).
5. Washing
(1) After incubation, aspirate the staining working solution from each well.
(2) Add Component C (1× Annexin V Binding Buffer) to wash cells using the same volume as in Step 2.
6. Microscopic Observation and Imaging
(1) After washing, add the corresponding volume of Component C (1× Annexin V Binding Buffer) to each well to keep cells moist.
(2) Place the plate directly under a fluorescence microscope for observation and imaging. Filter settings are the same as in Protocol A.
Protocol C: Flow Cytometry (for Suspension and Adherent Cells)
1. Collection and Preparation of Single-Cell Suspension
(1) Suspension cells: Same as Protocol A. Finally, collect 5×10⁵-1×10⁶ cells.
(2) Adherent cells:
a. Aspirate cell culture medium into a suitable centrifuge tube and reserve. Wash adherent cells briefly with a small amount of PBS, then transfer the washed PBS into the same tube containing the medium. Centrifuge at 1000 rpm for 5 min at room temperature.
b. Add an appropriate volume of EDTA-free trypsin to digest the cells.
Note: Trypsin volume, digestion time, and temperature follow laboratory standard protocols.
c. Critical step: Add the reserved cell culture medium from Step a to terminate digestion.
d. Transfer the cell suspension to an appropriate centrifuge tube, centrifuge at 1000 rpm for 5 min at room temperature, and aspirate the supernatant.
e. Combine cells from Step a and Step d.
f. Resuspend cells in PBS and perform cell counting.
g. Take 5×10⁴-1×10⁵ cells, centrifuge at 1000 rpm for 5 min at room temperature, and aspirate the supernatant.
h. Wash cells with 100 μL of Component C, centrifuge at 1000 rpm for 5 min at room temperature, and aspirate the supernatant.
Note: Trypsin digestion is critical for adherent cells. Insufficient digestion requires harsh pipetting to detach cells, causing mechanical damage and false-positive apoptosis. Excessive digestion also leads to false-positive apoptosis, impairs PS exposure on the cell membrane, interferes with Annexin V binding, and affects apoptosis detection.
2. Cell Staining
(1) Resuspend cells in 100 μL of Component C (1× Annexin V Binding Buffer) .
(2) Add 5 μL of Component A (LumiDye™ 488 Caspase-3/7 Substrate), 5 μL of Component B (LumiDye™ 647-Annexin V), 1 μL of Component E (Hoechst 33342), and 1 μL of Component F (EthD Gold). Mix gently by pipetting.
(3) Incubate at room temperature for 15-30 min in the dark (incubation time can be optimized based on preliminary experiments).
Note: Dye volumes can be adjusted according to cell type, preliminary tests, or experimental conditions, but the volume ratio of Component A (LumiDye™ 488 Caspase-3/7 Substrate) to Component D (Caspase-3/7 Inhibitor) must remain 5:1.
3. Washing and Resuspension
(1) After incubation, centrifuge at 1000 rpm for 5 min at room temperature to collect cells.
(2) Aspirate the supernatant, add 100 μL of Component C (1× Annexin V Binding Buffer), and gently resuspend for washing.
(3) Centrifuge again and aspirate the supernatant completely.
(4) Add 500 μL of Component C (1× Annexin V Binding Buffer) and gently resuspend cells to prepare the flow cytometry sample.
Note: Keep samples on ice before detection.
4. Instrument Detection
(1) Important: Perform flow cytometry within 1 h after staining to ensure stable fluorescence signals.
(2) Analyze on a flow cytometer using preset detection channels.
(3) First, adjust voltage using unstained cells (blank control) so that the cell population is located in the lower-left quadrant of the plot.
(4) Then run negative and positive controls to validate the experimental system.
(5) Finally, test experimental samples and record the mean fluorescence intensity for analysis.
LumiDye™ 488 Caspase-3/7 Substrate: FITC channel (Ex/Em: 490/525 nm)
LumiDye™ 647-Annexin V: APC channel (Ex/Em: 650/665 nm)
Hoechst 33342: DAPI channel (Ex/Em: 350/461 nm)
III. Result Interpretation
1. Qualitative Analysis (Microscopy)
Figure 1. Fluorescence microscopy images of Live Cell Caspase‑3/7 Activity and Annexin V Dual Apoptosis Detection Kit
(1) Negative group: Normal K562 cells co‑stained with LumiDye™ 488 Caspase‑3/7 Substrate, LumiDye™ 647‑Annexin V, EthD Gold, and Hoechst 33342, followed by merged imaging.
(2) Positive group: Apoptosis‑induced K562 cells co‑stained with LumiDye™ 488 Caspase‑3/7 Substrate, LumiDye™ 647‑Annexin V, EthD Gold, and Hoechst 33342, followed by merged imaging.
(3) Inhibited group: K562 cells treated with apoptosis induction plus Caspase‑3/7 inhibition, co‑stained with LumiDye™ 488 Caspase‑3/7 Substrate, LumiDye™ 647‑Annexin V, EthD Gold, and Hoechst 33342, followed by merged imaging.
Result Interpretation:
Compared with the negative control, apoptosis induction significantly increased Caspase‑3/7 activity (marked by enhanced green fluorescence intensity and cell count), elevated phosphatidylserine (PS) externalization (marked by enhanced red fluorescence intensity and cell count), and increased EthD Gold signal. These results indicate activated Caspase‑3/7 activity, prominent PS externalization, and an increased number of apoptotic cells.After treatment with Caspase‑3/7 Inhibitor, Caspase‑3/7 activity was markedly reduced compared with the positive group (decreased green fluorescence intensity and cell count), accompanied by significantly reduced PS externalization. However, PS externalization and apoptotic cell count in the inhibited group remained higher than those in the negative group, suggesting that alternative apoptotic pathways are activated following Caspase‑3/7 inhibition.
Note: Actual results may vary depending on detection instruments, operating conditions, cell types, and other factors. This example is for reference only.
Frequently Asked Questions and Answers
1. Q: Can cells be fixed before or after staining?
A: To observe different states of apoptotic cells, only live cells induced to undergo apoptosis can be used for detection. If cells are fixed, they will all die during fixation, resulting in numerous false-positive apoptotic signals in staining results.
2. Q: Why should the cell culture medium be retained when performing flow cytometry on adherent cells?
A: Apoptotic cells already exist after drug treatment. These cells gradually lose adherence ability and detach into the medium. These cells must be included during staining and detection; otherwise, the proportion of apoptotic or necrotic cells in results may be underestimated, leading to incorrect evaluation of drug or cellular treatment effects. In addition, serum contained in the culture medium can efficiently neutralize trypsin. Residual trypsin degrades Annexin V in subsequent staining, causing staining failure.
4. Q: Why did the Caspase-3/7 Inhibitor not suppress LumiDye™ 488 staining in my cells?
A: Caspase-3/7 Inhibitor is a reversible competitive inhibitor with limited cell permeability, which may be insufficient to block extremely high levels of Caspase-3/7 activity. In our experience, inhibitor treatment may reduce cellular fluorescence intensity from LumiDye™ staining, but usually not to the level of untreated control cells. Adding an irreversible inhibitor such as Z-DEVD-FMK before or after apoptosis induction may more effectively suppress caspase activity.
5. Q: Can cells be fixed after dual apoptosis analysis?
A: Yes. It is recommended to fix with 2-4% formaldehyde at room temperature for 10–15 min. Over-fixation may cause signal reduction. To preserve Annexin V staining, 1.25 mM CaCl₂ should be added to all buffers. Methanol fixation is not recommended. LumiDye™ 488 staining can withstand permeabilization with 0.1% Triton X-100 for subsequent immunostaining, although signal intensity may decrease after permeabilization and washing. Annexin V staining cannot withstand detergent permeabilization.
6. Q: How specific is the LumiDye™ Caspase-3/7 Substrate for Caspase-3/7?
A: Like other Caspase-3/7 substrates, the LumiDye™ Caspase-3/7 Substrate is based on the DEVD consensus sequence for Caspase-3/7, which can also be cleaved by Caspase-7. Due to overlapping substrate specificity among caspases, other caspases may also cleave the DEVD substrate.
7. Q: What cell types can be used with LumiDye™ 488 Caspase-3/7 Substrate?
A: LumiDye™ 488 Caspase-3/7 Substrate has been reported effective in various primary cells and immortalized cell lines in published scientific literature.
8. Q: Can dual apoptosis analysis be used for tissue staining?
A: Dual apoptosis analysis cannot be used for fixed cells or tissue sections. We have not validated this assay for live tissue staining.
ZJ26F0434541
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