细胞自噬/细胞毒性双色检测试剂盒 (MDC/PI法)，为细胞水平研究自噬与细胞毒性的调控机制提供便捷工具。该试剂盒以单丹磺酰尸胺 (MDC)作为自噬检测探针，以碘化丙啶 (PI)作为细胞死亡标志物，可实现两种指标的同步分析。
自噬是细胞重要的生理过程，具体是指细胞借助溶酶体对自身内部成分 (如细胞器、蛋白质等)进行降解与消化。这一过程不仅能帮助细胞高效调动、循环利用自身组分，还可阻止受损细胞器、错误折叠蛋白质及入侵微生物在细胞内积累。自噬的发生需经历多步反应：首先，细胞质中的待降解成分会被双层膜结构包裹，形成自噬体；随后，自噬体与溶酶体融合形成自噬溶酶体，内部的细胞物质最终在自噬溶酶体内完成降解。
在检测原理方面，MDC作为一种荧光化合物，可通过离子捕获机制与膜脂质相互作用，特异性富集到自噬体中，呈现绿色荧光，从而实现自噬的可视化检测。同时，试剂盒中的PI可用于细胞毒性分析，PI能对坏死细胞及凋亡晚期膜完整性受损的细胞进行染色，使这类细胞呈现红色荧光。需注意的是，MDC也可能标记其他酸性细胞器，且正常细胞会存在基础背景荧光，因此在结果分析时需设置对照组以确保准确性。
正常的自噬对细胞存活、分化、发育及内环境稳态至关重要，而自噬调控异常则与癌症、感染性疾病、衰老及退行性疾病密切相关。值得关注的是，自噬在应对应激时通常发挥促进细胞存活的作用，但在特定条件下也可能诱导细胞死亡；且自噬与细胞凋亡的关系十分复杂，二者不仅拥有共同的刺激因素和组成成分，还能相互调控彼此的活性。因此，深入研究细胞选择自噬或凋亡途径的具体影响因素及调控机制，具有重要的科研意义。
注：效能等同Abcam Autophagy/Cytotoxicity Dual Staining试剂盒 (ab133075)。
应用范围：药物、营养素、缺氧、氧化应激等条件对细胞自噬及细胞毒性的分析
产品特点：
1. 同步双检：一次实验即可检测细胞自噬与细胞毒性，便于探究二者关联；
2. 多设备适配：染色后可经荧光显微镜实时观察形态，或用酶标仪、流式细胞仪定量分析，满足不同检测需求；
3. 真实性：MDC对细胞毒性较低，能更接近生理状态下的自噬水平，避免固定或透化处理对细胞结构的破坏；
4. 实验快速：无需复杂预处理，10 min 染色流程简单，节省实验时间；
5. 操作简便：无需转染，直接对活细胞染色，无需任何转染或稳转株构建；
6. 结果直观：荧光颜色区分明确，易观察，且支持量化分析，数据清晰易解读。
产品参数
1. MDC Stain Ex/Em: 335/512 nm；
2. PI Ex/Em: 493/636 nm (without DNA)；
3. PI Ex/Em: 535/617 nm (with DNA)。
产品组分：
| C1520245 | 组件 | 100 T | 10×100 T | Storage | | C1520245A | 10× MDC染色液 | 1 mL | 10×1 mL | -20℃. Store in the dark. | | C1520245B | 染色缓冲液 | 50 mL | 10×50 mL | 2-8℃. | | C1520245C | 洗涤缓冲液 | 100 mL | 10×100 mL | 2-8℃. | | C1520245D | PI | 500 μL | 10×500 μL | -20℃. Store in the dark. |
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注：100 T为试剂盒组分A (10× MDC染色液)进行96孔板荧光显微镜检测贴壁细胞时可检测的微孔总数。
注意事项：
1. 荧光染料易受光照影响而淬灭，请务必避光操作，以保持染料稳定性。
2. 染料对人体有一定危害，实验人员必须穿实验服并佩戴一次性手套进行操作，必要时可佩戴护目镜等额外防护装备，避免试剂直接接触皮肤、黏膜及眼睛。
3. 操作过程中需尽量注意避光，同时减少所有试剂暴露于强光下的时间。
4. 实验中可通过设置正常细胞对照组，对比分析药物处理组与对照组的荧光强度，判断细胞自噬是否被诱导或抑制。同时，需注意排除非特异性染色的干扰，结合自噬相关指标进一步验证结果。
5. 本产品仅限于科研用途并且不得存放于普通住宅内。
6. 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：
将试剂盒中的组分A (10× MDC Stain)、组分B (Stain Buffer)、组分C (Wash Buffer)、组分D (PI)从储存条件 (如-20 ℃/4 °C)取出，平衡至室温 (15-25 °C)，准备足量PBS缓冲液。
2. 仪器准备：
荧光显微镜：MDC检测选择紫外激发滤光片330-350 nm；发射波长510-530 nm，发出绿色荧光；PI和DNA结合形成复合物后最大激发波长为535 nm；最大发射波长617 nm。
3. 对照组设置：
| 组别 | MDC | PI | 样品细胞 | | 空白对照 | - | - | 不做任何处理 | | 阴性对照 | + | + | 不做任何处理 | | 阳性对照 | + | + | 自噬诱导细胞 | | 实验组 | + | + | 实验组细胞 |
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(1) 空白对照：未染色细胞，用于评估细胞自发荧光。
(2) 阴性对照：未进行自噬诱导的染色细胞，作为基础自噬水平。
(3) 阳性对照：使用EBSS (Earle’sBalancedSalt Solution)饥饿处理细胞以诱导自噬，建议初次实验时在37 °C孵育30 min至 4 h并设置时间梯度，以确定最佳诱导时间。
(4) 实验组：根据实验设计，提前对细胞进行相应的处理 (如药物、饥饿处理等)。
注：阳性对照中EBSS缺乏氨基酸和血清，可模拟“饥饿状态”，是细胞自噬研究中常用的阳性对照诱导方法。
注：不同细胞系对自噬诱导的敏感性存在差异，请根据具体细胞类型优化处理时长。如部分非神经元细胞 (如HeLa、HEK293)在EBSS处理30 min后即可观察到自噬激活，但具体响应时间应结合实验检测手段评估。
二、 操作步骤
方案一：荧光显微镜检测 (适用于悬浮细胞)
3. 细胞重悬与计数：
(1) 加入适量组分C (洗涤缓冲液)重悬并细胞计数。
(2) 取5×10⁴-10⁵个细胞，1000 rpm低速离心5 min，去除组分C。
(3) 使用100 μL组分B (染色缓冲液)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约70%-85%为宜。
注：组分B (染色缓冲液)为我司自主创新的染色缓冲液，配方经多重优化，可在检测期间持续维持细胞生理状态并提供基础营养，并显著提升染料稳定性。若样本类型差异导致用量不足，可直接用PBS、HBSS等常规缓冲液补足体积重悬，不影响检测结果。
4. 细胞染色：
(1) 按组分A (10× MDC染色液): 组分B (染色缓冲液): 组分D (PI)=2: 17: 1的比例配置MDC/PI染色工作液，将染色液加入到细胞中 (现用现配、避光配置)。工作液配制见表1。
表1. MDC/PI 染色工作液配制
(按照96孔板每孔100 μl染色工作液体系，参考下表配制MDC/PI染色工作液，并充分混匀)
| 反应组分 | 1 Sample | 5 Samples | | A. 10× MDC染色液 | 10 μL | 50 μL | | B. 染色缓冲液 | 85 μL | 425 μL | | D. PI | 5 μL | 25 μL | | 总体积 | 100 μL | 500 μL |
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注：表 1. (配制反应混合物体积可按该比例根据具体情况而定)
(2) 向每孔加入100 μL MDC/PI染色工作液后，在37 ºC避光条件下孵育15 min。其中，孵育时长可根据实际观察到的染色效果进行灵活调整，调整区间为10-60 min。
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轻柔重悬细胞，进行洗涤。重复此洗涤步骤共3次，以充分去除未结合的游离染料，降低背景。
(4) 末次洗涤后，彻底吸净上清液，加入100 μL组分C重悬细胞，制成待测样品。
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。
(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，寻找并拍摄绿色点状或囊泡状荧光 (即自噬体/自噬溶酶体)。
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%-85%为最佳。
注：染色完成后必须立即对样品进行分析。建议先检测MDC染色情况，再检测PI染色情况，以避免MDC荧光在分析过程中耗竭。
方案二：荧光显微镜检测 (适用于贴壁细胞)
1. 细胞处理：
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. 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C (Wash Buffer)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：
根据样品孔数，按分A (10× MDC Stain): 组分B (Stain Buffer): 组分D (PI)=2: 17: 1的比例，提前在避光条件下配制MDC/PI染色工作液 (现用现配)，将染色液加入到细胞中。工作液配制见上表1。
4. 细胞染色：
(1) 向各孔中加入对应体积的MDC/PI 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250 μL/孔，12孔板加500 μL/孔，6孔板加1 mL/孔)。
(2) 向每孔加入100 μL MDC/PI染色工作液后，在37 ºC避光条件下，孵育15 min。其中，孵育时长可根据实际观察到的染色效果进行灵活调整，调整区间为10-60 min。
5. 洗涤：
(1) 孵育结束后，吸弃各孔中的染色工作液。
(2) 按步骤1的体积，加入组分C洗涤细胞。重复此洗涤步骤共3次，每次都要彻底吸净洗涤液。
6. 显微镜观察与拍照：
(1) 末次洗涤后，每孔加入对应体积的组分C，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。
注：染色完成后必须立即对样品进行分析。建议先检测MDC染色情况，再检测PI染色情况，以避免MDC荧光在分析过程中耗竭。
三、 结果判读：
定性分析 (显微镜)：
图 1.MDC 染色效果图
(1) 检测ZnMet是否能诱导成纤维细胞产生更多自噬体，设置空白对照组、Met 组、ZnCL2组、ZnMet组排除单一成分干扰。经MDC探针孵育并通过Nikon confocal A1R采集荧光成像。结果表明 ZnMet可激活自噬通路。
注：图片引自《A novel sprayable thermosensitive hydrogel coupled with zinc modified metformin promotes the healing of skinwound. Bioact Mater.》（doi: 10.1016/j.bioactmat.2022.06.008.）
注：MDC是含有氨基的酸性染料，MDC亦可标记其它酸性细胞器，正常细胞存在基础背景荧光，结果分析时需设对照。
(2) 结果判读 (推荐)
| 细胞状态 | 自噬探针荧光 (MDC，绿色) | 细胞毒性探针荧光 (PI，红色) | 结果说明 | | 正常活细胞 | 弱荧光/无荧光 | 无荧光 | 自噬水平低，细胞膜完整 | | 自噬激活活细胞 | 强荧光 (自噬体点状分布) | 无荧光 | 自噬水平高，细胞存活 | | 濒死/死细胞 | 弱荧光 / 无荧光 | 强荧光 (胞质弥漫分布) | 细胞膜受损，细胞毒性阳性 | | 自噬激活濒死细胞 | 强荧光 | 红色荧光 | 自噬激活但细胞已受损 |
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常见疑问与解答：
1. 问：染色后大量贴壁细胞脱落，该如何解决？
答：对贴壁能力弱的细胞，提前用多聚赖氨酸、鼠尾胶原或明胶预处理培养板，增强细胞与培养板的吸附力。操作时轻柔加样，避免直接冲击细胞层；洗涤时缓慢倾斜培养板，沿壁加入洗涤液，减少液体流动对细胞的扰动。
2. 问：MDC染色时间 (10-60 min)应该如何精确选择？
答：这是一个需要优化的参数。时间过短可能导致信号弱，时间过长则可能增加非特异性染色和背景。建议做法：在正式实验前，先用阳性对照细胞 (EBSS 饥饿处理)做一个预实验如30 min，再根据预实验结果调整染色时间在10-60 min范围内。通过显微镜观察或流式检测，选择信噪比最高 (即阳性信号最强，背景最低)的时间点作为后续实验的标准染色时间。
3. 问：为什么显微镜方案要洗涤3次？
答：显微镜：是直接成像，任何残留的游离染料都会在视野中形成均匀的背景荧光，严重干扰对点状自噬体信号的观察。因此需要严格洗涤 3 次，以最大化信噪比。
4. 问：阴性对照染色强度高，背景干扰大，该如何排查？
答：阴性对照染色强度高可能与操作步骤不规范 (如洗涤次数不足、染色时间过长)或阳性对照设置不当有关。建议严格遵循洗涤步骤，确保MDC染色后至少洗涤3次或更多，以彻底清除残留染料。同时，核对检测仪器参数，确保激发波长为350-380 nm，发射波长为512-532 nm；并设置阳性对照 (如经EBSS诱导30 min-4 h的细胞）和空白对照，通过对比验证阴性对照的可靠性。
5. 问：没有观察到预期的荧光，可能是什么原因？如何解决？
答：未观察到预期荧光可能与操作步骤错误、激发光设置不正确或阳性对照设置不当有关。建议仔细核对并确保激发光波长正确，严格按照操作步骤执行实验，并确认阳性对照的设置是否合理 (如使用EBSS诱导的细胞)。
6. 问：MDC和PI染色荧光强度均较低，可能是什么原因？
答：可能的原因是细胞密度过低；细胞在处理过程中丢失。
Autophagy/Cytotoxicity Dual Detection Kit (MDC/PI Method) provides a convenient tool for studying the regulatory mechanisms of autophagy and cytotoxicity at the cellular level. Using monodansylcadaverine (MDC) as an autophagy probe and propidium iodide (PI) as a cell death marker, this kit enables simultaneous analysis of both indicators.
Autophagy is a vital physiological process in which cells degrade and digest intracellular components (such as organelles and proteins) via lysosomes. This process not only allows cells to efficiently mobilize and recycle their own constituents, but also prevents the accumulation of damaged organelles, misfolded proteins, and invasive microorganisms. Autophagy proceeds through multiple steps: first, cytoplasmic components to be degraded are enclosed by a double-membrane structure to form autophagosomes; autophagosomes then fuse with lysosomes to form autolysosomes, where intracellular materials are ultimately degraded.
Regarding the detection principle: as a fluorescent compound, MDC interacts with membrane lipids through an ion-trapping mechanism and specifically accumulates in autophagosomes, emitting green fluorescence for the visual detection of autophagy. Meanwhile, PI in the kit is used for cytotoxicity analysis. PI stains necrotic cells and late apoptotic cells with impaired membrane integrity, causing these cells to emit red fluorescence. Note that MDC may also label other acidic organelles, and normal cells exhibit basal background fluorescence. Therefore, control groups should be included in result analysis to ensure accuracy.
Normal autophagy is essential for cell survival, differentiation, development, and homeostasis, while dysregulated autophagy is closely associated with cancer, infectious diseases, aging, and degenerative diseases. Notably, autophagy generally promotes cell survival under stress, but may also induce cell death under specific conditions. The relationship between autophagy and apoptosis is highly complex: they share common stimuli and components, and can mutually regulate each other’s activity. Thus, in-depth research into the factors and regulatory mechanisms governing cell fate decisions between autophagy and apoptosis is of great scientific significance.
Note: Performance equivalent to Abcam Autophagy/Cytotoxicity Dual Staining Kit (ab133075).
Applications
Analysis of cellular autophagy and cytotoxicity under conditions including drugs, nutrients, hypoxia, and oxidative stress.
Product Features
1. Dual simultaneous detection: Autophagy and cytotoxicity can be measured in one assay, facilitating investigation of their relationship.
2. Multi-instrument compatible: Stained samples can be observed in real time by fluorescence microscopy for morphology, or quantitatively analyzed by microplate reader and flow cytometry to meet diverse detection requirements.
3. Physiological relevance: MDC has low cytotoxicity, allowing detection closer to physiological autophagy levels and avoiding structural damage from fixation or permeabilization.
4. Rapid assay: No complicated pretreatment; staining is completed in 10 minutes with a simple procedure, saving experimental time.
5. Easy operation: No transfection required; direct staining of living cells without transfection or stable cell line construction.
6. Intuitive results: Clear distinction by fluorescence color for easy observation, with support for quantitative analysis and interpretable data.
Product Parameters
1. MDC Stain Ex/Em: 335/512 nm
2. PI Ex/Em: 493/636 nm (free, without DNA)
3. PI Ex/Em: 535/617 nm (DNA-bound)
Kit Components
| C1520245 | Component | 100 T | 10×100 T | Storage | | C1520245A | 10× MDC Stain | 1 mL | 10×1 mL | -20℃. Store in the dark. | | C1520245B | Stain Buffer | 50 mL | 10×50 mL | 2-8℃. | | C1520245C | Wash Buffer | 100 mL | 10×100 mL | 2-8℃. | | C1520245D | PI | 500 μL | 10×500 μL | -20℃. Store in the dark. |
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Note: 100 T refers to the total number of wells detectable for adherent cells using Component A (10× MDC Stain) in a 96-well plate under fluorescence microscopy.
Precautions
1. Fluorescent dyes are susceptible to quenching upon light exposure. Please perform all operations in the dark to maintain dye stability.
2. The dyes are potentially harmful to humans. Personnel must wear lab coats and disposable gloves during operation. Additional protective equipment such as goggles may be worn if necessary to avoid direct contact of reagents with skin, mucous membranes and eyes.
3. Keep away from light during operation and minimize the time that all reagents are exposed to strong light.
4. Normal cell control groups can be set up in the experiment to compare and analyze the fluorescence intensity between the drug-treated group and the control group, so as to determine whether autophagy is induced or inhibited. Meanwhile, interference from non-specific staining should be excluded, and results should be further verified in combination with autophagy-related indicators.
5. This product is for research use only and must not be stored in ordinary residential premises.
6. For your safety and health, please follow the standard laboratory safety regulations of your institution.
Instructions for Use
I. Pre-Experiment Preparation
1. Reagent Preparation
Remove Component A (10× MDC Stain), Component B (Stain Buffer), Component C (Wash Buffer), and Component D (PI) from storage (e.g., -20 °C/4 °C) and equilibrate to room temperature (15-25 °C). Prepare an adequate volume of PBS buffer.
2. Instrument Preparation
Fluorescence microscope:
For MDC detection: Use an ultraviolet excitation filter (330–350 nm) and an emission filter (510–530 nm) to detect green fluorescence.
For PI: After binding to DNA, the maximum excitation wavelength is 535 nm and the maximum emission wavelength is 617 nm.
3. Control Group Setup
| Group | MDC | PI | Sample Cell | | Blank Control | - | - | Untreated cells | | Negative Control | + | + | Untreated cells | | Positive Control | + | + | Autophagy-induced cells | | Experimental Group | + | + | Experimentally treated cells |
|
(1) Blank Control: Unstained cells, used to assess cellular autofluorescence.
(2) Negative Control: Stained cells without autophagy induction, representing the basal autophagy level.
(3) Positive Control: Induce autophagy by starving cells in EBSS (Earle’s Balanced Salt Solution). For initial experiments, incubate at 37 °C for 30 min to 4 h with a time gradient to determine the optimal induction duration.
(4) Experimental Group: Treat cells in advance according to the experimental design (e.g., drug treatment, starvation, etc.).
Note: EBSS lacks amino acids and serum, mimicking a "starvation state"-it is a commonly used positive control induction method in autophagy research.
Note: Sensitivity to autophagy induction varies across cell lines; optimize the treatment duration for your specific cell type. For example, some non-neuronal cells (e.g., HeLa, HEK293) show autophagy activation after 30 min of EBSS treatment, but the exact response time should be evaluated using your experimental detection method.
II. Procedure
Protocol A: Fluorescence Microscopy (For Suspension Cells)
3. Cell Resuspension and Counting
(1) Add an appropriate volume of Component C (Stain Buffer) to resuspend the cells and perform cell counting.
(2) Take 5×10⁴-1×10⁵ cells, centrifuge at 1000 rpm for 5 min at low speed, and remove Component C.
(3) Resuspend in 100 μL of Component B (Wash Buffer). Centrifuge at 1000 rpm for 5 min at low speed and discard the supernatant.
Note: The microscopy method is not strict with cell density, as long as the density is consistent between the experimental group and the control group. It can be flexibly adjusted according to sample type and experimental conditions, with a recommended cell coverage of 70%-85% under the microscope.
Note: Component B (Stain Buffer) is an independently developed staining buffer optimized through multiple formulations. It maintains cell physiological status and provides basic nutrition during detection, while significantly improving dye stability. If volume is insufficient due to sample differences, PBS, HBSS or other conventional buffers may be used to adjust the volume for resuspension without affecting results.
4. Cell Staining
(1) Prepare MDC/PI staining working solution at a ratio of Component A (10× MDC Stain): Component B (Stain Buffer): Component D (PI)=2: 17: 1, then add the working solution to the cells. (Prepare fresh before use and protect from light during preparation.) See Table 1 for working solution preparation.
Table 1. Preparation of MDC/PI Staining Working Solution
(For a 100 μL staining system per well in a 96‑well plate, prepare MDC/PI staining working solution according to the table below and mix thoroughly.)
| Reaction Component | 1 Sample | 5 Samples | | A. 10× MDC Stain | 10 μL | 50 μL | | B. Stain Buffer | 85 μL | 425 μL | | D. PI | 5 μL | 25 μL | | Total Volume | 100 μL | 500 μL |
|
Note: The volume of the reaction mixture can be scaled proportionally based on actual experimental needs.
(2) After adding 100 μL of MDC/PI staining working solution to each well, incubate at 37 ºC for 15 min in the dark. The incubation time can be flexibly adjusted from 10 to 60 min according to the actual staining effect observed.
5. Washing and Resuspension
(1) After incubation, centrifuge at 1000 rpm for 5 min at room temperature to collect the cells.
(2) Carefully aspirate and discard the dye-containing supernatant (this is waste liquid and should be disposed of properly).
(3) Add 100 μL of Component C and gently resuspend the cells for washing. Repeat this washing step 3 times to sufficiently remove unbound free dye and reduce background.
(4) After the final wash, completely aspirate the supernatant, add 100 μL of Component C to resuspend the cells, and prepare the sample for testing.
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 the cells naturally settle to the bottom, then observe under a fluorescence microscope.
(2) Slide method: Pipette 20-30 μL of cell suspension onto a clean glass slide, gently cover with a coverslip to avoid air bubbles, and observe under a fluorescence microscope.
(3) Under preset filter conditions, locate and capture green punctate or vesicular fluorescence (i.e., autophagosomes/autolysosomes).
Note: The volume of cell suspension can be adjusted according to cell count. Optimal cell distribution in the microscope field is 70%-85%.
Note: Samples must be analyzed immediately after staining. It is recommended to detect MDC staining first, followed by PI staining, to avoid depletion of MDC fluorescence during analysis.
Protocol B: Fluorescence Microscopy (For Adherent Cells)
1. Cell Treatment
Inoculate cells into a plate one day in advance to reach 70%-85% confluence. After cell attachment, treat cells according to the experimental design.
2. Cell Washing
(1) Aspirate and discard the cell culture medium directly from the culture plate.
(2) Add an appropriate volume of Component C (Wash Buffer) according to the plate format to gently wash the cells once (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). Completely aspirate Component C after washing.
3. Preparation of Staining Working SolutionBased on the number of sample wells, prepare MDC/PI staining working solution in advance at a ratio of Component A (10× MDC Stain): Component B (Stain Buffer): Component D (PI)=2: 17: 1 under dark conditions (prepare fresh before use), then add the working solution to the cells. See Table 1 above for preparation.
4. Cell Staining
(1) Add the corresponding volume of MDC/PI staining working solution to each well (same volume as in Step 2, 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) After adding 100 μL of MDC/PI staining working solution to each well, incubate at 37 ºC for 15 min in the dark. The incubation time can be flexibly adjusted from 10 to 60 min according to the actual staining effect observed.
5. Washing
(1) After incubation, aspirate and discard the staining working solution from each well.
(2) Add Component C to wash the cells at the same volume as in Step 1. Repeat this washing step 3 times, and completely aspirate the wash buffer each time.
6. Microscopic Observation and Imaging
(1) After the final wash, add the corresponding volume of Component C to each well to keep the cells moist.
(2) Observe and image the culture plate directly under a fluorescence microscope. Filter settings are the same as in Protocol A.
Note: Samples must be analyzed immediately after staining. It is recommended to detect MDC staining first, followed by PI staining, to avoid depletion of MDC fluorescence during analysis.
III. Result Interpretation
Qualitative Analysis (Microscopy):
Figure 1. MDC Staining Image
(1) To detect whether ZnMet can induce more autophagosomes in fibroblasts, a blank control group, Met group, ZnCl₂ group, and ZnMet group were set up to exclude interference from single components. After incubation with MDC probe, fluorescence images were captured using a Nikon confocal A1R system. The results indicated that ZnMet can activate the autophagy pathway.
Note: Image reproduced from A novel sprayable thermosensitive hydrogel coupled with zinc modified metformin promotes the healing of skin wound. Bioact Mater. (doi: 10.1016/j.bioactmat.2022.06.008.)
Note: MDC is an acidic dye containing an amino group. MDC may also label other acidic organelles, and normal cells exhibit basal background fluorescence. Therefore, control groups must be included for result analysis.
(2) Result Interpretation (Recommended)
| Cell Status | Autophagy Probe Fluorescence (MDC, Green) | Cytotoxicity Probe Fluorescence (PI, Red) | Result Description | | Normal Viable Cells | Weak/No fluorescence | No fluorescence | Low autophagy level, intact cell membrane | | Autophagy-Activated Viable Cells | Strong fluorescence (punctate autophagosome distribution) | No fluorescence | High autophagy level, viable cells | | Dying/Dead Cells | Weak/No fluorescence | Strong fluorescence (diffuse cytoplasmic distribution) | Damaged cell membrane, positive cytotoxicity | | Autophagy-Activated Dying Cells | Strong fluorescence | Red fluorescence | Autophagy activated but cells damaged |
|
Frequently Asked Questions
1. Q: A large number of adherent cells detach after staining. How to solve this problem?
A: For cells with weak adherence, pre-treat the culture plate with poly-L-lysine, rat tail collagen, or gelatin in advance to enhance cell attachment to the plate. Add samples gently during operation to avoid direct impact on the cell layer. Tilt the plate slowly during washing and add washing buffer along the wall to reduce disturbance to cells by liquid flow.
2. Q: How to precisely select the MDC staining time (10-60 min)?
A: This is a parameter that requires optimization. Too short a time may result in weak signals, while too long a time may increase non-specific staining and background. Recommendation: Before formal experiments, perform a preliminary experiment using positive control cells (EBSS starvation treatment), e.g., 30 min, then adjust the staining time within 10-60 min according to the preliminary results. Select the time point with the highest signal-to-noise ratio (strongest positive signal, lowest background) via microscopy or flow cytometry as the standard staining time for subsequent experiments.
3. Q: Why wash 3 times in the microscopy protocol?
A: Microscopy involves direct imaging. Any residual free dye will produce uniform background fluorescence in the field of view, severely interfering with the observation of punctate autophagosome signals. Therefore, strict washing 3 times is required to maximize the signal-to-noise ratio.
4. Q: How to troubleshoot high staining intensity and strong background in the negative control?
A: High background in the negative control may result from non-standard procedures (e.g., insufficient washing times, excessive staining duration) or inappropriate positive control setup. Strictly follow the washing steps to ensure at least 3 washes after MDC staining to thoroughly remove residual dye. Meanwhile, verify instrument parameters to ensure excitation wavelength is 350–380 nm and emission wavelength is 512-532 nm. Set up positive controls (e.g., cells induced with EBSS for 30 min-4 h) and blank controls to verify the reliability of the negative control by comparison.
5. Q: Why is the expected fluorescence not observed? How to resolve it?
A: Failure to observe expected fluorescence may result from incorrect procedures, improper excitation light settings, or inappropriate positive control setup. Carefully verify correct excitation wavelength settings, strictly follow the experimental protocol, and confirm reasonable positive control setup (e.g., using EBSS-induced cells).
6. Q: What causes low fluorescence intensity of both MDC and PI staining?
A: Possible causes are excessively low cell density or cell loss during processing.
ZJ26F0535133
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