该试剂盒是一款集成化细胞状态检测工具，通过“磷脂酰丝氨酸 (PS)外翻+膜完整性评估+全细胞核染色”的多参数荧光标记策略，实现健康细胞、凋亡细胞与坏死细胞的同步区分及量化分析。其核心检测原理清晰互补：
(1) 针对凋亡细胞的PS外翻特性，采用高亮度、光稳定性强的LumiDye™ 488标记Annexin V，在钙离子介导下与细胞表面暴露的PS特异性结合，使凋亡细胞呈现绿色荧光，同时提供适配基础研究的FITC及适合多色分析的R-PE/APC标记选项；
(2) 基于膜完整性差异，坏死及晚期凋亡细胞的细胞膜破损，允许高电荷核酸探针EthD Gold进入并结合DNA，产生红色荧光，还提供适配基础研究7-AAD；
(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；
相较于传统单一标记试剂盒，本品优势显著：多参数同步检测避免样本分批次处理的误差，流式细胞仪象限分析可直观区分细胞类型 (Q3：健康细胞、Q4：早期凋亡细胞、Q1：晚期凋亡细胞、Q2：坏死细胞)，且兼容荧光显微镜下的形态学观察，尤其适合动态追踪凋亡进程，量化凋亡细胞、坏死细胞在全细胞群落中所占比例。其应用覆盖细胞生理状态基础研究、抗癌药/抗菌肽的疗效评估、肿瘤细胞凋亡机制解析及临床样本中细胞状态的辅助检测等场景。
细胞凋亡与坏死作为两类截然不同的细胞死亡方式，其精准区分对生命科学研究与临床转化具有不可替代的价值。凋亡是主动有序的“细胞自杀”过程，参与胚胎发育、组织稳态维持及免疫清除，其过度激活或抑制与癌症、阿尔茨海默病等重大疾病密切相关；坏死则由急性损伤触发，伴随炎症反应，是脓毒症、心肌梗死等疾病的核心病理特征；而健康细胞的存活状态直接反映代谢功能与信号通路完整性，是药物筛选的核心评估指标。传统检测工具常因单一标记局限，难以同步获取三类细胞的量化数据，导致研究效率受限。本试剂盒的创新性在于突破这一壁垒，通过多参数协同的检测逻辑，在同一体系中实现细胞状态的标准化、精准化分析，不仅为揭示凋亡/坏死平衡失调的分子机制 (如线粒体通路调控)提供可靠工具，更能支撑药物诱导细胞死亡效率的量化评估，同时为临床样本检测 (如肿瘤预后判断、感染性疾病诊断)提供有力的技术支撑，助力基础研究向临床应用的高效转化。
应用范围：贴壁细胞或悬浮细胞中凋亡细胞、坏死细胞、健康细胞检测；肿瘤研究、药物毒性评价、干细胞分化监控等领域，尤其适合动态追踪凋亡进程 (如化疗药物诱导的细胞死亡模式)；
产品特点：
1. 全状态同步检测：一次实验覆盖凋亡、坏死、健康细胞状态，避免传统单一标记局限性，实现“三合一”精准区分；
2. 高效省时：整体染色时间仅需15 min，远快于传统多步检测流程，大幅提升实验效率染色时间；
3. 荧光强度优异：LumiDye™ 488荧光量子产率高，光稳定性强，确保凋亡细胞信号清晰；
4. 高特异性与灵敏度：EthD Gold比PI或EthD-I更强的DNA结合亲和力和荧光量子产率，减少假阳性；
5. 操作简便性：一步染色即可完成多参数检测，无需复杂预处理，结果可直接通过流式散点图或显微图像量化；
产品参数：
1. Annexin V-APC Ex/Em: 650/660 nm；
2. 7-AAD Ex/Em: 549/648 nm；
3. Hoechst 33342 Ex/Em: 350/461 nm (with DNA)。
产品组分：
| 项目号 | 物理外观 | 组件 | 10 T | 50 T | 100 T | Storage | | E1520231A | 液体 | 1× Annexin V结合缓冲液 | 5 mL | 50 mL×2 | 50 mL×4 | 2-8℃. Store in the dark. | | E1520231B | 液体 | Annexin V-APC | 50 μL | 250 μL | 500 μL | 2-8℃. Store in the dark. | | E1520231C | 液体 | 7-AAD | 10 μL | 50 μL | 100 μL | 2-8℃. Store in the dark. | | E1520231D | 液体 | Hoechst 33342 | 10 μL | 50 μL | 100 μL | 2-8℃. Store in the dark. |
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注：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. 本实验中使用的细胞必须是未固定的细胞，Annexin V-APC等均依赖于正常细胞中的完整细胞膜进行区分健康细胞与凋亡或坏死细胞。
2. 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。
3. 本产品仅限于科研用途并且不得存放于普通住宅内。
4. 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 (15-25 °C)，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：490/515 nm、Ex/Em：532/625 nm、Ex/Em：350/461 nm、Ex/Em：494/518nm、Ex/Em：488/578 nm、Ex/Em：549/648 nm、Ex/Em：599~644/678~697 nm、Ex/Em：650/660 nm；
(2) 流式细胞仪：检测通道激发/发射波长Ex/Em：490/515 nm、Ex/Em：532/625 nm、Ex/Em：350/461 nm、Ex/Em：494/518nm、Ex/Em：488/578 nm、Ex/Em：549/648 nm、Ex/Em：599~644/678~697 nm、Ex/Em：650/660 nm。
3. 对照组设置：
| 组别 | Annexin V-APC | 7-AAD | Hoechst 33342 | 样品细胞 | | 空白对照 | - | - | - | 不做任何处理 | | 阴性对照 | + | + | + | 不做任何处理 | | 单阳对照 1 | + | - | - | 诱导剂处理后明显凋亡 | | 单阳对照 2 | - | + | - | 诱导剂处理后明显凋亡 | | 单阳对照 3 | - | - | + | 诱导剂处理后明显凋亡 | | 实验组 | + | + | + | 实验组细胞 |
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(1) 空白对照组：评估背景荧光干扰、检测细胞自身的自发荧光、流式检测中用于调节阈值和电压。
(2) 阴性对照组：排除染料的非特异性结合，验证染色的特异性。
(3) 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。
4. 细胞准备：
根据实验设计，对细胞进行相应的药物处理。
二、 操作步骤
方案一：荧光显微镜检测 (适用于悬浮细胞)
1. 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) 加入100 μL PBS轻柔重悬细胞，进行洗涤。
(4) 再次1000 rpm，室温离心5 min，彻底吸弃上清液。
2. 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A (1×Annexin V Binding Buffer)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 (推荐范围：10⁵-10⁷ cells/mL)。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。
3. 细胞染色：
(1) 取100 μL组分A重悬细胞，分别加入组分B (Annexin V-APC)5 μL、组分C (7-AAD)1 μL和组分D (Hoechet 33342)1 μL，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)。
4. 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A轻柔重悬细胞，进行洗涤。
(4) 末次洗涤后，彻底吸净上清液，加入100 μL组分A重悬细胞，制成待测样品。
5. 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。
(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) 在预设的滤光片条件下，寻找细胞并分别拍摄。
Annexin V-APC 检测早期凋亡信号：选用APC通道 (Ex/Em：650/660 nm)或相应滤光片；
7-AAD检测坏死细胞信号：选用PI或Texas Red通道 (Ex/Em：550/650 nm)或相应滤光片；
Hoechet 33342检测健康细胞信号：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. 细胞清洗：
(1) 直接在培养板中吸弃细胞培养液。
(2) 根据培养板规格，加入对应体积的组分A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A。
2. 制备染色工作液：
根据样品孔数，取适量体积的组分A，按照每100 μL中三种染料分别为组分B：5 μL、组分C：1 μL和组分D：1 μL的使用量将染料加入到组分A中，混匀备用 (现用现配)。例如，为1个96孔板染色，需配制10.7 mL工作液 (10 mL组分A+500 μL组分B+100 μL组分+100 μL组分D)。
3. 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 (2)，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)。
(2) 室温避光孵育15-30 min (孵育时间可根据预实验结果优化)。
4. 洗涤：
(1) 孵育结束后，吸弃各孔中的染色工作液。
(2) 按步骤1 (2)的体积，加入组分A (1×Annexin V Binding Buffer)洗涤细胞，清洗后吸去清洗缓冲液。
5. 显微镜观察与拍照：
(1) 洗涤后，每孔加入对应体积的组分A，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。
Annexin V-APC检测早期凋亡信号：选用APC通道 (Ex/Em：650/660 nm)或相应滤光片；
7-AAD检测坏死细胞信号：选用PI或Texas Red通道 (Ex/Em：550/650 nm)或相应滤光片；
Hoechet 33342检测健康细胞信号：选用DAPI通道 (Ex/Em：350/461 nm)或相应滤光片。
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“方案一”的步骤1和2，最终，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 (覆盖细胞即可)，室温下置于显微镜下观察，待细胞变圆、间隙增大时，用移液器轻轻吹打，使细胞完全脱落。
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。
c. 关键：加入“a 步骤”离心管内备用的细胞培养基，终止消化。
注：离心管内备用的细胞培养基非常重要，不能直接丢弃，其中会含有已经凋亡或坏死的目的细胞，且细胞培养基中的血清能够有效终止胰酶的消化作用，否则残留胰酶将会严重影响后续的凋亡检测。
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A清洗细胞，1000 rpm，室温离心5 min，吸弃上清。
2. 细胞染色：
(1) 用100 μL组分A重悬细胞。
(2) 加入组分B (Annexin V-APC)5 μL、组分C (7-AAD)1 μL和组分D (Hoechet 33342)1 μL。
(3) 室温避光孵育15-30 min (孵育时间可根据预实验结果优化)。
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A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轻柔重悬细胞，制成流式上机样品。
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。
Annexin V-APC检测早期凋亡信号：选用APC通道 (Ex/Em：650/660 nm)
7-AAD检测坏死细胞信号：选用PC5.5通道 (Ex/Em：550/650 nm)
Hoechet 33342检测有核细胞信号：选用DAPI通道 (Ex/Em：350/461 nm)
三、 结果判读：
1. 定性分析 (显微镜)：
图1. 一体化凋亡、坏死和健康细胞全状态加强版检测试剂盒 (Annexin V-APC, 7-AAD, Hoechst 33342)(E1520231)荧光镜检拍照
(1) 阴性对照：K562正常细胞，进行Annexin V-APC (A)、7-AAD (B)、Hoechet 33342 (C)共染后合成画面 (D)；
(2) 阳性对照：诱导凋亡处理后K562细胞，Annexin V-APC (E)、7-AAD (F)、Hoechet 33342 (G)共染合成画面 (H)。
结果判读：
(1) 早期凋亡细胞：细胞膜磷脂酰丝氨酸外翻 (PS)显著 (Annexin V-APC远红荧光信号强)；
(2) 膜破损的凋亡晚期细胞：Annexin V-APC远红荧光+7-AAD红色荧光+Hoechet 33342蓝色荧光；
(3) 坏死细胞：仅7-AAD红色荧光信号；
(4) 健康细胞：红/远红荧光双阴性，蓝色细胞核荧光均匀染色。
在经过凋亡诱导处理后，与阴性对照相比PS外翻水平显著增加 (远红荧光亮度、细胞个数显著增强)，表明细胞膜PS外翻显著。晚期凋亡细胞比例明显增加 (远红荧光/红色荧光强度/蓝色荧光、三阳细胞个数显著增多)，表明药物促进了细胞的凋亡进而导致细胞死亡。
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。
2. 定量分析 (流式细胞仪)：
以FSC-SSC构建散点图，来确定细胞群，再以Hoechst 33342-SSC构建散点图，排除细胞碎片；以Annexin V-APC为X轴，7-AAD为Y轴，将完整细胞群分为4个群体：
图2. 一体化凋亡、坏死和健康细胞全状态加强版检测试剂盒 (Annexin V-APC, 7-AAD, Hoechst 33342)(E1520231)流式细胞术分析
本实验以K562细胞为样本，以凋亡诱导组为阳性处理组 (图2.A)，以未处理组为阴性对照 (图2.B)，对K562细胞处理后染色，进行流式上机检测。
结果判读1：以图2.A为例：可以区分出健康细胞 (左下)、早期凋亡细胞 (右下)、晚期凋亡 (右上)、坏死细胞 (左上)。
结果判读2：对K562细胞进行凋亡诱导后，凋亡细胞的比例显著高于阴性对照；健康细胞比例显著低于阴性对照 (见图2.A、图2.B)。
通过比较阳性组与阴性组在一、四象限的占比，分析药物或者基因是促进细胞凋亡还抑制凋亡。数据大于对照组就是促进凋亡，小于对照组则为抑制凋亡。
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：要观察凋亡细胞坏死细胞和活细胞这三种细胞在同一细胞群中的状态，只能用活细胞。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坏死细胞的信号。
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或坏死细胞，这些细胞的贴壁能力逐渐降低，会游离在培养基中，染色检测时需要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AnnexinV，导致早期凋亡染色失败，出现假阴性结果。
3. 问：为什么检测时细胞总数量与预期相差较大，晚期凋亡和坏死细胞占比远远低于预期？
答：凋亡诱导或者药物处理后的细胞，进入凋亡阶段或者坏死之后，细胞形态与结构会和正常细胞产生差异，特别是坏死细胞可能由于细胞膜破裂内容物弥散，与正常细胞差异巨大；在实验中进行细胞离心和清洗过程中容易保留在上清中被丢弃，可能导致最终的细胞总数远少于预期，晚期凋亡和坏死细胞数量少于预期；可以在细胞离心后保留少量上清，避免吸走疏松的细胞沉淀。
4. 问：同样作为死细胞染料，为什么选择EthD Gold而不选择PI进行标记？
答：EthD Gold在与PI具有相同的染色结果的基础上，表现出更具有选择性的优势：
a. 亮度更高：与DNA结合后荧光强度比EthD-I再提升45%，同等曝光下信号更强，拍照或流式检测更敏感。
b. 特异性更好：分子量大、膜通透性要求更严格，仅标记膜严重破损的细胞，不标记“轻度膜渗漏”的晚期凋亡，减少假阳性。
c. 毒性更低：工作浓度可比PI低3-7倍即可达到同等或更强荧光，长时间孵育几乎不引起额外细胞死亡，流式坏死群比例更接近真实。
d. 无RNA干扰：几乎不与RNA结合，无需额外RNase处理，DNA群像更干净，流式直方图CV值更小。
e. 分群更锐利：双参数图上EthD Gold阳性群与阴性群间距大，早期/晚期/坏死三种界限清晰，便于gate设定。
f. 兼容固定前检测：虽同样不可固定，但在“活染-上机”流程中信号稳定，固定前拍照/采集不会出现PI常见的洗脱-扩散问题。
This kit is an integrated cellular status detection tool. It adopts a multi-parameter fluorescent labeling strategy including phosphatidylserine (PS) externalization, membrane integrity assessment and total nuclear staining, to realize simultaneous differentiation and quantitative analysis of viable, apoptotic and necrotic cells. Its complementary and clear detection principles are as follows:
(1) Based on PS externalization characteristic of apoptotic cells, Annexin V is labeled with high-brightness and photostable LumiDye™ 488. It specifically binds to PS exposed on cell surface mediated by calcium ions, rendering apoptotic cells green fluorescence. The kit also provides FITC labeling for basic research and R-PE/APC labeling for multicolor flow cytometry analysis.
(2) According to differences in cell membrane integrity, damaged membranes of necrotic and late apoptotic cells allow high-charge nucleic acid probe EthD Gold to penetrate and bind DNA, producing red fluorescence. Alternative dyes including 7-AAD for basic research are also available.
(3) Hoechst 33342 blue dye penetrates all cells universally and stains cell nuclei, serving as a stable internal reference for cell counting and quantitative calibration.
Compared with traditional single-label kits, this product has outstanding advantages. Multi-parameter simultaneous detection avoids errors caused by batch sample processing. Quadrant analysis by flow cytometry clearly distinguishes cell subsets: Q3: viable cells, Q4: early apoptotic cells, Q1: late apoptotic cells, Q2: necrotic cells. It is also compatible with morphological observation under fluorescence microscope, especially suitable for dynamic tracing of apoptotic progression and quantifying the proportion of apoptotic and necrotic cells in total cell population. Applications include basic research on cellular physiological status, efficacy evaluation of anticancer drugs and antimicrobial peptides, mechanism analysis of tumor apoptosis, and auxiliary detection of cellular status in clinical samples.
Apoptosis and necrosis are two distinct modes of cell death. Accurate discrimination between them is irreplaceable for life science research and clinical translation. Apoptosis is an active and orderly programmed cell death process involved in embryonic development, tissue homeostasis maintenance and immune clearance. Abnormal activation or inhibition of apoptosis is closely associated with cancers, Alzheimer’s disease and other critical illnesses. Necrosis is triggered by acute injury accompanied by inflammatory response, acting as a core pathological feature of sepsis, myocardial infarction and other diseases. Viable cell status directly reflects metabolic function and signal pathway integrity, serving as a key indicator for drug screening. Traditional single-label assays cannot simultaneously obtain quantitative data of three cell types, limiting research efficiency. This kit breaks such limitations. With synergistic multi-parameter detection, it achieves standardized and accurate cellular status analysis in one system. It provides reliable tools for revealing molecular mechanisms of apoptosis-necrosis imbalance (such as mitochondrial pathway regulation), supports quantitative assessment of drug-induced cell death efficiency, and offers technical support for clinical sample detection including tumor prognosis and infectious disease diagnosis, facilitating efficient translation from basic research to clinical application.
Applications
Detection of viable, apoptotic and necrotic cells in adherent and suspension cells; tumor research, drug toxicity assessment, stem cell differentiation monitoring. Particularly suitable for dynamic tracing of apoptotic processes such as chemotherapy-induced cell death patterns.
Product Features
1. Simultaneous detection of full cell status: One experiment distinguishes viable, apoptotic and necrotic cells, overcoming limitations of traditional single labeling and realizing three-in-one accurate classification.
2. Time-saving and high efficiency: Total staining procedure only takes 15 minutes, much faster than conventional multi-step protocols, greatly improving experimental throughput.
3. Excellent fluorescence intensity: LumiDye™ 488 possesses high quantum yield and superior photostability, ensuring clear apoptotic cell signals.
4. High specificity and sensitivity: EthD Gold exhibits stronger DNA-binding affinity and fluorescence quantum yield than PI or EthD-I, reducing false positive results.
5. Simple operation: Multi-parameter detection is completed by one-step staining without complicated pretreatment. Results can be directly quantified via flow scatter plots or microscopic images.
Product Specifications
1. Annexin V-APC Ex/Em: 650/660 nm
2. 7-AAD Ex/Em: 549/648 nm
3. Hoechst 33342 Ex/Em: 350/461 nm (bound to DNA)
Kit Components
| Item No. | Appearance | Components | 10 T | 50 T | 100 T | Storage | | E1520231A | Liquid | 1× Annexin V Binding Buffer | 5 mL | 50 mL×2 | 50 mL×4 | 2-8℃. Store in the dark. | | E1520231B | Liquid | Annexin V-APC | 50 μL | 250 μL | 500 μL | 2-8℃. Store in the dark. | | E1520231C | Liquid | 7-AAD | 10 μL | 50 μL | 100 μL | 2-8℃. Store in the dark. | | E1520231D | Liquid | Hoechst 33342 | 10 μL | 50 μL | 100 μL | 2-8℃. Store in the dark. |
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Precautions
1. Unfixed live cells are required for this assay. Annexin V-APC rely on intact cell membranes to differentiate viable, apoptotic and necrotic cells.
2. Single-staining control groups are recommended for fluorescence compensation adjustment in flow cytometry.
3. For research use only. Do not store this product in residential environments.
4. Comply with routine laboratory safety regulations to protect personal safety and health.
Instructions for Use
I. Pre-experiment Preparation
1. Reagent PreparationEquilibrate all kit components to room temperature (15–25 °C) and prepare sufficient PBS buffer.
2. Instrument Preparation
(1) Fluorescence microscope: Compatible excitation/emission wavelengths: 490/515 nm, 532/625 nm, 350/461 nm, 494/518 nm, 488/578 nm, 549/648 nm, 599~644/678~697 nm, 650/660 nm.
(2) Flow cytometer: Detection channels match the wavelengths above accordingly.
3. Control Group Setup
| Group | Annexin V-APC | 7-AAD | Hoechst 33342 | Sample Cells | | Blank Control | - | - | - | Untreated cells | | Negative Control | + | + | + | Untreated cells | | Single Positive Control 1 | + | - | - | Cells with obvious apoptosis after inducer treatment | | Single Positive Control 2 | - | + | - | Cells with obvious apoptosis after inducer treatment | | Single Positive Control 3 | - | - | + | Cells with obvious apoptosis after inducer treatment | | Test Group | + | + | + | Test sample cells |
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(1) Blank control: Evaluate background fluorescence and cellular autofluorescence, adjust threshold and voltage in flow cytometry.
(2) Negative control: Eliminate non-specific dye binding and verify staining specificity.
(3) Single-positive control: Avoid fluorescence spillover, calibrate signal intensity and adjust fluorescence compensation.
4. Cell Preparation
Treat cells with corresponding drugs according to experimental design.
II. Assay Procedures
Protocol 1: Fluorescence Microscopy (Suspension Cells)
1. Cell Collection and Washing
(1) Transfer suspended cell suspension into centrifuge tubes.
(2) Centrifuge at 1000 rpm for 5 min at room temperature and carefully discard supernatant.
(3) Resuspend cells gently with 100 μL PBS and wash.
(4) Centrifuge again at 1000 rpm for 5 min at room temperature and completely remove supernatant.
2. Cell Resuspension and Counting
(1) Resuspend cells with PBS and perform cell counting.
(2) Take 5×10⁴–1×10⁵ cells, centrifuge at low speed 1000 rpm for 5 min and remove PBS.
(3) Resuspend cells in 100 μL Buffer A (1×Annexin V Binding Buffer), centrifuge and discard supernatant.
Note: Cell density is flexible for microscopic detection as long as groups are consistent (recommended: 10⁵–10⁷ cells/mL). Adjust appropriately to ensure clear cell distribution under microscope.
3. Cell Staining
(1) To 100 μL Buffer A-resuspended cells, add 5 μL Buffer B (Annexin V-APC), 1 μL Buffer C (7-AAD) and 1 μL Buffer D (Hoechst 33342). Mix gently by pipetting.
(2) Incubate for 15–30 min at room temperature in dark. Incubation time can be optimized via preliminary tests.
4. Washing and Resuspension
(1) After incubation, centrifuge cells at 1000 rpm for 5 min at room temperature.
(2) Discard dye-containing supernatant properly as hazardous waste.
(3) Wash cells with 100 μL Buffer A.
(4) After final washing, remove supernatant completely and resuspend cells in 100 μL Buffer A for detection.
5. Microscopic Observation and Imaging
(1) 96-well plate method: Add 100 μL cell suspension into wells, let cells settle naturally, then observe under fluorescence microscope.
(2) Slide method: Drop 20–30 μL cell suspension onto clean slides, cover coverslips carefully to avoid bubbles.
(3) Capture images under corresponding filter channels:
Annexin V-APC (early apoptosis): APC channel, Ex/Em 650/660 nm
7-AAD (necrosis): PI/Texas Red channel, Ex/Em 550/650 nm
Hoechst 33342 (viable nucleus): DAPI channel, Ex/Em 350/461 nm
Protocol 2: Fluorescence Microscopy (Adherent Cells)
1. Cell Washing
(1) Discard culture medium directly from culture plates.
(2) Wash cells with Buffer A at appropriate volume: 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. Remove Buffer A after washing.
2. Prepare Staining Working Solution
According to well quantity, mix Buffer A with dyes freshly before use: 5 μL Buffer B, 1 μL Buffer C, 1 μL Buffer D per 100 μL Buffer A.Example: Staining one 96-well plate requires 10.7 mL working solution (10 mL A + 500 μL B + 100 μL C + 100 μL D).
3. Cell Staining
(1) Add an appropriate volume of staining working solution to each well (same volume as Step 1 (2): 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 for 15–30 minutes at room temperature in the dark. The incubation time can be optimized according to preliminary experimental results.
4. Washing
(1) Discard staining solution after incubation.
(2) Wash cells with Buffer A and remove washing buffer.
5. Microscopic Observation
(1) Add fresh Buffer A to keep cells moist.
(2) Observe and image plates directly under microscope with identical filter settings as Protocol 1.
Protocol 3: Flow Cytometry (Suspension & Adherent Cells)
1. Single Cell Suspension Preparation
(1) Suspension cells: Follow Protocol 1 Steps 1–2, collect 5×10⁵–1×10⁶ cells.
(2) Adherent cells:
a. Collect culture medium into centrifuge tubes. Rinse adherent cells with PBS, combine washing PBS with medium and centrifuge at 1000 rpm for 5 min.
b. Add EDTA-free trypsin to cover cells. Observe under microscope; blow cells gently when cells round up and separate.
Note: Improper trypsin digestion causes mechanical damage or artificial apoptosis, interfering PS-Annexin V binding and leading to false results.
c. Terminate digestion with reserved culture medium containing serum.
d. Centrifuge cell suspension and discard supernatant.
e. Combine all harvested cells.
f. Resuspend and count cells with PBS.
g. Take 5×10⁴–1×10⁵ cells, centrifuge and remove supernatant.
h. Wash with 100 μL Buffer A, centrifuge and discard supernatant.
2. Cell Staining
(1) Resuspend cells in 100 μL Buffer A.
(2) Add 5 μL Annexin V-APC, 1 μL 7-AAD and 1 μL Hoechst 33342.
(3) Incubate 15–30 min at room temperature in dark.
3. Washing and Resuspension
(1) Centrifuge cells after incubation.
(2) Wash once with Buffer A.
(3) Remove supernatant completely.
(4) Resuspend cells in 500 μL Buffer A for flow detection.
4. Flow Detection
(1) Detect samples within 1 hour after staining to maintain stable fluorescence.
(2) Analyze on flow cytometer with preset channels.
(3) Adjust voltage using unstained blank control to locate cell population at bottom-left quadrant.
(4) Verify assay validity with negative and positive controls.
(5) Detect samples and record mean fluorescence intensity.
III. Result Interpretation
1. Qualitative Analysis (Microscopy)
Figure 1 All-in-One Apoptotic, Necrotic and Healthy Cell Comprehensive Status Enhanced Detection Kit (Annexin V-APC, 7-AAD, Hoechst 33342) (E1520231)
(1) Negative control: Normal K562 cells co-stained with Annexin V-APC, 7-AAD and Hoechst 33342.
(2) Positive control: Apoptosis-induced K562 cells with triple staining.
Interpretation Criteria:
(1) Early apoptotic cells: Strong Annexin V-APC far-red fluorescence with significant PS externalization
(2) Late apoptotic cells: Double positive for Annexin V-APC and 7-AAD, plus Hoechst blue nuclear signal
(3) Necrotic cells: Only 7-AAD red fluorescence
(4) Viable cells: Double negative for red/far-red fluorescence, uniform blue nuclear staining
Apoptosis-induced groups show markedly increased PS externalization and higher proportion of late apoptotic cells, indicating drug-induced cell apoptosis. Results may vary by instruments, operation and cell types; images are for reference only.
2. Quantitative Analysis (Flow Cytometry)
Gate cell population via FSC-SSC scatter plot, exclude debris by Hoechst 33342-SSC. Divide intact cells into 4 quadrants using Annexin V-APC (X-axis) and 7-AAD (Y-axis).
Figure 2 Flow cytometry profiles of E1520231 kit
In this assay, K562 cells were used as samples. The apoptosis-induced group served as the positive treatment group (Figure 2.A), and the untreated group served as the negative control (Figure 2.B). K562 cells were stained after treatment and detected by flow cytometry.
Interpretation 1: Taking Figure 2.A as an example, viable cells (lower left), early apoptotic cells (lower right), late apoptotic cells (upper right) and necrotic cells (upper left) can be clearly distinguished.
Interpretation 2: After apoptosis induction in K562 cells, the proportion of apoptotic cells was significantly higher than that in the negative control, while the proportion of viable cells was markedly lower (see Figure 2.A and Figure 2.B).
By comparing the proportions of cells in quadrants Q1 and Q4 between the positive group and negative control group, we can determine whether drugs or genes promote or inhibit cell apoptosis. A higher proportion than the control indicates pro-apoptotic effect, while a lower proportion indicates anti-apoptotic effect.
FAQ
1. Q: Can fixed cells be used before or after staining?
A: Only live unfixed cells are applicable. Cell fixation causes artificial death and massive false positive apoptotic/necrotic signals.
2. Q: Why retain original culture medium for adherent cell flow assay?
A: Apoptotic/necrotic cells detach into medium and must be included for accurate ratio calculation. Serum in medium neutralizes trypsin to avoid Annexin V degradation and false negative results.
3. Q: Why total cell count and late apoptotic/necrotic proportion are lower than expected?
A: Damaged apoptotic/necrotic cells are easily lost in supernatant during centrifugation. Reserve small volume of supernatant to preserve loose cell pellets.
4. Q: Why EthD Gold is selected instead of PI as a dead cell stain?
A: While achieving consistent staining performance with PI, EthD Gold exhibits superior selectivity advantages:
a. Higher brightness: Its fluorescence intensity upon DNA binding is 45% higher than EthD-I. It delivers stronger signals under identical exposure conditions, offering higher sensitivity for microscopic imaging and flow cytometry detection.
b. Better specificity: With a larger molecular weight and stricter membrane permeability requirements, it only labels cells with severely damaged membranes, excluding late apoptotic cells with mild membrane leakage, thus reducing false positive results.
c. Lower cytotoxicity: It produces equivalent or stronger fluorescence at 3–7 times lower working concentration than PI. Prolonged incubation barely induces additional cell death, making the necrotic cell ratio detected by flow cytometry more authentic.
d. No RNA interference: It hardly binds to RNA, eliminating the need for additional RNase treatment. It produces cleaner DNA cell populations and lower CV values in flow histograms.
e. Sharper population separation: EthD Gold shows clear separation between positive and negative populations on dual-parameter plots. Boundaries among early apoptosis, late apoptosis and necrosis are distinct, facilitating convenient gating.
f. Compatible with pre-fixation detection: Although it cannot be fixed either, it maintains stable signals during live staining and flow detection. Unlike PI, it avoids common signal elution and diffusion issues during imaging and data acquisition before cell fixation.
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