是否无菌：是
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，采用高亮度的R-PE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，使凋亡细胞呈现红色荧光；
(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Draq 7；
(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：R-PE荧光量子产率高，确保凋亡细胞信号清晰；
4. 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-R-PE Ex/Em: 488/578 nm；
2. Draq 7 Ex/Em: 599~644/678~697 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 | | E1520241A | 液体 | 1× Annexin V结合缓冲液 | 5 mL | 50 mL×2 | 50 mL×4 | 2-8℃. Store in the dark. | | E1520241B | 液体 | Annexin V-R-PE | 50 μL | 250 μL | 500 μL | 2-8℃. Store in the dark. | | E1520241C | 液体 | Draq 7 | 10 μL | 50 μL | 100 μL | 2-8℃. Store in the dark. | | E1520241D | 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-R-PE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：Ex/Em：488/578 nm、Ex/Em：599~644/678~697 nm、Ex/Em：350/461 nm；
(2) 流式细胞仪：检测通道激发/发射波长Ex/Em：Ex/Em：488/578 nm、Ex/Em：599~644/678~697 nm、Ex/Em：350/461 nm。
3. 对照组设置：
| 组别 | Annexin V-R-PE
| Draq 7
| 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 |
|
(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 (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-R-PE)5 μL、组分C (Draq 7)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-R-PE检测早期凋亡信号：Ex/Em：488/578 nm；
Draq 7检测坏死细胞信号：Ex/Em：599~644/678~697 nm；
Hoechet 33342检测健康细胞信号：Ex/Em：350/461 nm。
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C+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结合缓冲液)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-R-PE检测早期凋亡信号：Ex/Em：488/578 nm；
Draq 7检测坏死细胞信号：Ex/Em：599~644/678~697 nm；
Hoechet 33342检测健康细胞信号：Ex/Em：350/461 nm。
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-R-PE)5 μL、组分C (Draq 7)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-R-PE检测早期凋亡信号：Ex/Em：488/578 nm；
Draq 7检测坏死细胞信号：Ex/Em：599~644/678~697 nm；
Hoechet 33342检测健康细胞信号：Ex/Em：350/461 nm。
三、 结果判读：
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-R-PE, Draq 7, Hoechst 33342)荧光镜检拍照
K562细胞阴性对照组正常培养作为对照；阳性对照组加入诱导剂CCCP，16h后使用试剂盒进行染色后荧光显微镜检测结果，为了荧光颜色明显区分PE荧光在显微镜信号采集系统中显示时赋色为绿色，DRAQ7赋色为红色。
(1) 阴性对照：K562正常细胞，进行Annexin V-R-PE、Draq 7、Hoechet 33342共染后合成画面；
(2) 阳性对照：诱导凋亡处理后K562细胞，Annexin V-R-PE、Draq 7、Hoechet 33342共染合成画面。
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-R-PE红色荧光信号强)；
(2) 膜破损的凋亡晚期细胞：Annexin V-R-PE红色荧光+Draq 7远红荧光+Hoechet 33342蓝色荧光；
(3) 坏死细胞：仅Draq 7远红荧光信号；
(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，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。
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。
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) Targeting the phosphatidylserine (PS) externalization characteristic of apoptotic cells, Annexin V is labeled with high-brightness R-PE. It specifically binds to PS exposed on the cell surface in a calcium-dependent manner, enabling apoptotic cells to exhibit red fluorescence.
(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 Draq 7 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. 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-R-PE Ex/Em: 488/578 nm；
2. Draq 7 Ex/Em: 599~644/678~697 nm；
3. Hoechst 33342 Ex/Em: 350/461 nm (with DNA)。
Kit Components
Item No.
| Appearance | Components | 10 T | 50 T | 100 T | Storage | | E1520241A | Liquid | 1× Annexin V Binding Buffer | 5 mL | 50 mL×2 | 50 mL×4 | 2-8℃. Store in the dark. | | E1520241B | Liquid | Annexin V-R-PE | 50 μL | 250 μL | 500 μL | 2-8℃. Store in the dark. | | E1520241C | Liquid | Draq 7 | 10 μL | 50 μL | 100 μL | 2-8℃. Store in the dark. | | E1520241D | Liquid | Hoechst 33342 | 10 μL | 50 μL | 100 μL | 2-8℃. Store in the dark. |
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Note: The number of uses of the kit refers to the total number of microwells that can be tested when performing fluorescence microscopy assays with a 96-well plate.
Precautions
1. Unfixed live cells are required for this assay. Annexin V-R-PE 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: Excitation/Emission wavelength Ex/Em: 488/578 nm, Ex/Em: 599~644/678~697 nm, Ex/Em: 350/461 nm;
(2) Flow cytometer: Detection channel excitation/emission wavelength Ex/Em: 488/578 nm, Ex/Em: 599~644/678~697 nm, Ex/Em: 350/461 nm.
3. Control Group Setup
| Group | Annexin V-R-PE
| Draq 7
| 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-R-PE), 1 μL Buffer C (Draq 7) 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-R-PE for early apoptosis detection: Ex/Em: 488/578 nm;
DRAQ7 for necrotic cell detection: Ex/Em: 599~644/678~697 nm;
Hoechst 33342 for viable cell detection: 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) After incubation, aspirate and discard the staining working solution in each well.
(2) Add Component A (1× Annexin V Binding Buffer) to wash cells at the volume specified in Step 1 (2). Aspirate the washing buffer after rinsing.
5. Microscopic Observation and Imaging
(1) After washing, add an appropriate volume of Component A to each well to keep cells moist.
(2) Place the culture plate directly under a fluorescence microscope for observation and image capture. The filter setting is the same as Protocol Ⅰ.
Annexin V-R-PE detects early apoptotic signals: Ex/Em: 488/578 nm;
DRAQ7 detects necrotic cell signals: Ex/Em: 599~644/678~697 nm;
Hoechst 33342 detects viable cell signals: Ex/Em: 350/461 nm.
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. Aspirate the cell culture medium into a suitable centrifuge tube for later use. Rinse adherent cells once with a small amount of PBS; aspirate the rinsed PBS and transfer it into the same centrifuge tube containing the collected medium. Centrifuge at 1000 rpm for 5 minutes at room temperature.
b. Add an appropriate amount of EDTA-free trypsin digestion solution (just enough to cover the cells). Observe under a microscope at room temperature. When the cells become rounded and intercellular gaps widen, gently pipette up and down to completely detach the cells.
Note: Trypsin digestion is critical for adherent cells. Insufficient digestion time requires vigorous pipetting to detach cells, which may cause mechanical damage and lead to false-positive apoptosis results. Excessively long digestion time also induces false-positive apoptosis, affecting the binding of phosphatidylserine (PS) on the cell membrane to Annexin V and interfering with apoptosis detection.
c. Key step: Add the reserved cell culture medium from step a to terminate digestion.
Note: The reserved culture medium in the centrifuge tube must not be discarded. It contains apoptotic and necrotic target cells, and the serum in the medium can effectively neutralize trypsin activity. Residual trypsin will severely interfere with subsequent apoptosis detection if not terminated properly.
d. Transfer the cell suspension into a suitable centrifuge tube, centrifuge at 1000 rpm for 5 minutes at room temperature, then aspirate and discard the supernatant.
e. Collect all cells obtained from step a and step d.
f. Resuspend the cells with PBS and perform cell counting.
g. Harvest 5×10⁴ to 1×10⁵ cells, centrifuge at 1000 rpm for 5 minutes at room temperature, then aspirate and discard the supernatant.
h. Wash the cells with 100 μL of Component A, centrifuge at 1000 rpm for 5 minutes at room temperature, then aspirate and discard the supernatant.
2. Cell Staining
(1) Resuspend the cells with 100 μL of Component A.
(2) Add 5 μL of Component B (Annexin V-R-PE), 1 μL of Component C (DRAQ7) and 1 μL of Component D (Hoechst 33342).
(3) Incubate at room temperature for 15–30 min in the dark (the incubation time can be optimized according to the preliminary experimental results).
3. Washing and Resuspension
(1) After incubation, centrifuge at 1000 rpm at room temperature for 5 min to collect cells.
(2) Aspirate and discard the supernatant, add 100 μL of Component A, and resuspend gently for washing.
(3) Centrifuge again, and completely aspirate off the supernatant.
(4) Add 500 μL of Component C to gently resuspend the cells to prepare samples for flow cytometry detection.
4. On-board Detection
(1) Important: Perform flow cytometry detection within 1 hour after staining to ensure stable fluorescence signals.
(2) Analyze samples using a flow cytometer with preset detection channels.
(3) First adjust the voltage with blank control (unstained cells) to locate the cell population at the lower-left corner of the coordinate system.
(4) Then detect the negative control and positive control to confirm the validity of the experimental system.
(5) Finally test the experimental samples, and record and analyze the mean fluorescence intensity of cells.
Annexin V-R-PE for early apoptotic signal detection: Ex/Em: 488/578 nm;
DRAQ7 for necrotic cell signal detection: Ex/Em: 599~644/678~697 nm;
Hoechst 33342 for viable cell signal detection: Ex/Em: 350/461 nm.
III. Result Interpretation
Qualitative Analysis (Microscopy)
Figure 1 All-in-One Apoptotic, Necrotic and Healthy Cell Comprehensive Status Enhanced Detection Kit (Annexin V-R-PE, Draq 7, Hoechst 33342) fluorescence microscopic imaging
The negative control group of K562 cells was cultured under normal conditions as the control; the positive control group was treated with the apoptosis inducer CCCP. After 16 hours, cells were stained with the kit and observed under a fluorescence microscope. To clearly distinguish fluorescent signals, PE fluorescence was pseudo-colored green and DRAQ7 fluorescence pseudo-colored red in the microscopic signal acquisition system.
(1) Negative control: Normal K562 cells, composite imaging after co-staining with Annexin V-R-PE, DRAQ7 and Hoechst 33342;
(2) Positive control: Apoptosis-induced K562 cells, composite imaging after co-staining with Annexin V-R-PE, DRAQ7 and Hoechst 33342.
Result Interpretation
(1) Early apoptotic cells: Significant externalization of cell membrane phosphatidylserine (PS) with strong red fluorescent signal of Annexin V-R-PE;
(2) Late apoptotic cells with damaged membrane: Positive for Annexin V-R-PE red fluorescence, DRAQ7 far-red fluorescence and Hoechst 33342 blue fluorescence;
(3) Necrotic cells: Only positive for DRAQ7 far-red fluorescent signal;
(4) Viable cells: Double negative for red/far-red fluorescence, with uniform blue nuclear fluorescence staining.
After apoptosis induction, the level of PS externalization increased significantly compared with the negative control, indicating obvious PS eversion on the cell membrane. The proportion of late apoptotic cells was markedly elevated (significantly increased intensity of far-red/red/blue fluorescence and number of triple-positive cells), suggesting that the inducer promoted cell apoptosis and further led to cell death.
Note: Actual test results may vary due to differences in testing instruments, operating conditions, cell lines and other factors. This example is for reference only.
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.
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