DRAQ5是一款远红外亲脂性膜透性DNA染料，核心通过特异性结合DNA实现细胞核染色，可同时适用于活细胞与固定后死细胞检测。相较于传统细胞核染料，如Hoechst、DAPI这样最常用的蓝色荧光探针，激发波段为紫外，这两种染料就限制使用者不能与其他紫外激发或者显蓝色的荧光染料在同一实验中使用，DRAQ5的发射光位于远红外区域，完美避开紫外激发与蓝色荧光通道，解决了多通路检测中染料光谱重叠的限制。同时DRAQ5特异性结合DNA而不结合RNA，可用于细胞周期中DNA含量的分析。与PI用于细胞周期分析相比，无需RNase酶解、细胞破膜及洗涤步骤，大幅简化实验流程，为研究者节省操作时间并减少样本损耗。
DRAQ5凭借独特的光谱特性与便捷的操作优势，成为多色荧光成像和细胞周期分析的关键工具，应用包括：与GFP等荧光蛋白或其他可见光染料联合的多通路细胞标记，实现细胞结构与特定蛋白的共定位观察；直接用于融合GFP荧光蛋白细胞的增殖检测，通过核复染精准分析DNA含量；高效完成细胞周期进程研究，无需复杂前处理即可获得准确的DNA含量数据等。该染料的出现突破了传统染色技术的应用局限，为活细胞动态观察、高通量细胞分析等研究提供了灵活、高效的解决方案，推动细胞水平研究的便捷化与精准化。仅限体外科研用途 不得用于临床诊断或治疗。
注：效能等同于AAT ReadiUse™ DRAQ5 Staining Solution *5 mM in Water*（17558）。
注：本文中DRAQ5是Biostatus Limited的商标及注册商标；本文中ReadiUse™是 AAT Bioquest的商标及注册商标。
应用范围：
活细胞成像，活细胞实时核标记与动态观察，细胞周期分析，活细胞的DNA标记，染色质超微结构研究等；
产品特点：
1、免破膜/洗涤：活细胞直接染色，无需破膜处理，染色后无需洗涤；
2、极速染色：室温最快5 min快速染色，大幅缩短实验时间；
3、方便使用：以易用的5 mM液体形式提供，按需稀释后即用；
4、适合多色搭配：广谱的激发波段及远红外发射波段，适合多色搭配；
5、细胞成像：DRAQ5为膜透性细胞核染料，可用于活细胞成像以及核酸含量测定。
产品参数：
1、Ex/Em：601/699 nm；
2、Cas号：252903-95-0；
3、分子式：C₂₂H₂₈N₄O₄；
4、分子量：412.48；
5、光谱图：
产品组分：
| 组分 | 40 T | 100 T | 400 T | | Ready-to-use DRAQ5 Live Cell DNA Dye(5 mM in Water) | 20 μL | 50 μL | 200 μL |
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注：规格是以5×10⁴ cells/well为例，染色工作液浓度20 μM计算。
注意事项：
1、DRAQ5属于远红外荧光，肉眼不可见，需要CCD相机或激光共聚焦观察。
2、由于DRAQ5发射和激发波长范围很宽，不建议将DRAQ5与其他可被488 nm或633 nm激光激发的远红光荧光染料联用。
3、荧光染料存在淬灭问题，请尽量注意避光，以减缓淬灭。
4、使用前请将产品瞬时离心，再进行后续操作。
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：
(1) 将试剂从储存条件 (如4 °C)取出，平衡至室温 (15-25 °C)。
(2) 准备细胞培养基、缓冲液 (如不含叠氮化钠的PBS)。
2. 仪器准备：
荧光显微镜：配备633 nm或647 nm光源的荧光显微镜。
二、 操作步骤
方案一：荧光显微镜检测
1. 细胞准备：
准备不含有叠氮化钠的PBS缓冲液或特定细胞的特定培养基。用 PBS 或培养基重悬细胞，控制细胞密度≤4×10⁵ 个/mL。针对贴壁细胞或者组织则大致预估细胞数量。
2. 染色工作液制备：
根据表1稀释DRAQ5荧光染色液。DRAQ5染色液可以直接添加到贴壁细胞或组织切片表面，亦可直接添加到新鲜培养基中。
注：(1) 不同细胞种类所需DRAQ5的染色浓度可能不同，初期最好进行细胞染色的预实验工作，设置不同Draq5 染色浓度梯度确定最佳染色浓度。
(2) 当实验需要连续监测，如EGFP标记蛋白的易位实验时，DRAQ5染色液需要在加入任何激动剂/拮抗剂前 (通常是 0.5-3 h)以1 μM浓度加入。
表 1 细胞数目及所需DRAQ5体积及终浓度
| 细胞样品准备 | 细胞样品准备 | DRAQ5加入的体积及终浓度 | DRAQ5加入的体积及终浓度 | DRAQ5加入的体积及终浓度 | | 细胞数目 | PBS 或培养基体积 | 5 μM | 10 μM | 20 μM | | 1×10⁶ | 2500 μL | 2.5 μL | 5 μL | 10 μL | | 4×10⁵ | 1000 μL | 1 μL | 2 μL | 4 μL | | 2×10⁵ | 500 μL | 0.5 μL | 1 μL | 2 μL | | 1×10⁵ | 250 μL | 0.25 μL | 0.5 μL | 1 μL | | 5×10⁴ | 125 μL | 0.13 μL | 0.25 μL | 0.5 μL |
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3. 细胞染色
轻柔混匀，室温孵育5-30 min。孵育完成后无需清洗步骤。
4. 检测：
(1) 在流式细胞仪下检测，在488 nm处激发这种染料时，可使用685LP二向分色镜和710/50通道进行检测；在633nm处激发时，可以使用660/20通道进行检测。对于细胞周期/DNA分析应用，建议使用波长较长的滤光片，例如735LP二向分色镜和780/60通道来优化G1和G2/M峰的CV值。
(2) 在荧光显微镜下检测。建议使用633 nm或647 nm的光源进行激发。
三、 结果判读
定性分析（显微镜）：
图 1. DRAQ5 染料用于活细胞 DNA 染色的荧光显微成像
1、正常细胞核染色：活细胞核区呈现均匀红色荧光，核区边界清晰；
2、背景干扰：若细胞外或背景荧光强，提示洗涤不充分或染料浓度过高；
注：图片引自参考文献《DNA labeling in living cells》（doi: 10.1002/cyto.a.20172）
常见疑问与解答：
1、问：DRAQ5储液是否含DMSO？
答：该产品为水溶液形式，不含DMSO。
2、问：用DRAQ5对小鼠细胞进行2-3 h的活细胞成像。在对DRAQ5染色的细胞成像多久之后，在细胞毒性产生并使细胞死亡之前可以停止成像？
答：使用说明中有相应描述“DRAQ5染色在37 °C下加速，孵育时间 (5-30 min)可能会缩短”，但这应该通过滴定和每种细胞类型进行分别测定。在未添加任何激动剂/拮抗剂之前，可以在1 μM的检测持续时间 (通常为0.5-3 h)将DRAQ5添加到检测培养基中。DRAQ5染色发生得非常快，因此建议在分析前将DRAQ5添加到活细胞中。细胞的孵育时间不应超过3 h，但可能会因细胞系而异。
3、问：使用DRAQ5对上皮癌细胞的细胞核进行染色，发现荧光随着时间的推移变得越来越强。这种影响在激光功率较高和受热时发生得更强烈。这可能是什么原因呢？随着时间的推移，越来越多的染料与细胞核的DNA结合吗？它是否仅在与核DNA反应时发出荧光？是否知道染料在细胞凋亡过程中的行为。当染料变少时，染料能否与细胞核的DNA结合得一样好？
答：可以确认DRAQ5染色在较高温度下增强，因此可能需要调整孵育期。孵育期越长，我们预期的信号就越多，直到DNA饱和。如果DRAQ5随后被清洗，我们预计染色会随着时间的推移而缓慢下降。
除非DRAQ5与dsDNA结合，否则它几乎不会发出荧光。只有当它嵌入dsDNA中时，它才会产生强烈的荧光信号。DRAQ5染色在细胞凋亡中不受影响，因为DRAQ5对dsDNA的亲和力在活细胞或死细胞中没有差异。随着细胞凋亡中dsDNA的数量发生变化，DRAQ5信号的强度也会随着dsDNA的变化而变化。
4、问：正在进行18 h的细胞成像实验。在12 h后将DRAQ5培养基更换为不含DRAQ5的培养基时，染色强度会下降。
1）是否需要始终将DRAQ5保存在培养基中，即使可能会有毒性影响？
2）如果从培养基中去除DRAQ5，染色会持续多长时间？
答：a.我们预计细胞染色在培养基更换后会减少。尽管 DRAQ5对dsDNA 具有高亲和力，但仍存在平衡，去除培养基中的低残留浓度会破坏这种平衡。
b.我们认为染色在去除后可能会持续 1-2 h，但这可能取决于细胞暴露于先前使用的浓度。因为如果有任何毒性证据，人们会希望保持较低的浓度 (可能是 0.5-1 μM)。可以肯定的是，DRAQ5是否会产生毒性影响完全取决于细胞系和状态。根据文献报道可以合理地预测，未处于增殖状态的细胞对任何细胞通透性 DNA 嵌入探针的敏感性较低。例如，它可能更有可能适用于巨噬细胞或人脐静脉内皮细胞等终末分化细胞类型，其中它和密切相关的CyTRAKOrange已在长期实验中得到证明。如果使用的细胞系在使用时没有毒性表现，那么我们可以在DRAQ5存在的条件下持续进行 18 h 的实验。
5、问：在使用DRAQ5和DRAQ7的时候，这两者有什么区别，该如何选择？
答：对活细胞进行DNA 分析、长期监测细胞状态或进行组织深层成像时选DRAQ5；评估细胞活力、检测凋亡或区分样本中的死活细胞群体时选DRAQ7。两者的区别如下：
| 对比 | DRAQ5 | DRAQ7 | | 细胞膜渗透性 | 可透过 | 不可透过 | | 染色对象 | 活细胞和固定细胞的所有细胞核 | 仅死亡细胞、凋亡细胞或透化细胞的细胞核 | | 结合方式 | 与DNA化学计量结合，荧光强度反映DNA含量 | 特异性结合双链DNA，染色快速 | | 适用场景 | 活细胞DNA含量分析、细胞周期研究、长期活细胞成像实验、深层组织/肿瘤组织成像 | 细胞活力评估、细胞凋亡检测、药物/化合物体外毒性测试、区分死活细胞的流式细胞术实验 |
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DRAQ5 is a far-red lipophilic and membrane-permeable DNA dye that specifically binds to DNA for nuclear staining, and is suitable for the detection of both live cells and fixed dead cells. Compared with traditional nuclear dyes (e.g., Hoechst and DAPI, the most commonly used blue fluorescent probes) which are excited in the ultraviolet (UV) region, these two dyes restrict users from co-using them with other UV-excited or blue-emitting fluorescent dyes in the same experiment. DRAQ5 emits fluorescence in the far-red region, which perfectly avoids the UV excitation and blue fluorescence channels, thus solving the limitation of dye spectral overlap in multi-channel detection. Meanwhile, DRAQ5 specifically binds to DNA but not RNA, and can be used for the analysis of DNA content during the cell cycle. In comparison with Propidium Iodide (PI) for cell cycle analysis, DRAQ5 eliminates the need for RNase digestion, cell membrane lysis and washing steps, greatly simplifying the experimental workflow, saving operational time for researchers and reducing sample loss.
With its unique spectral properties and convenient operational advantages, DRAQ5 has become a key tool for multicolor fluorescence imaging and cell cycle analysis. Its applications include: multi-channel cell labeling in combination with fluorescent proteins (e.g., GFP) or other visible light dyes to realize colocalization observation of cell structures and specific proteins; direct use for proliferation detection of cells fused with GFP fluorescent proteins, with accurate analysis of DNA content via nuclear counterstaining; efficient study of cell cycle progression to obtain accurate DNA content data without complex preprocessing. The advent of this dye has broken the application limitations of traditional staining technologies, providing a flexible and efficient solution for research such as dynamic observation of live cells and high-throughput cell analysis, and promoting the convenience and accuracy of cell-level research. For in vitro research use only. Not for clinical diagnosis or treatment.
Note: Performance equivalent to AAT ReadiUse™ DRAQ5 Staining Solution 5 mM in Water (17558).
Note: DRAQ5 in this document is a trademark and registered trademark of Biostatus Limited; ReadiUse™ in this document is a trademark and registered trademark of AAT Bioquest.
Application Scope
Live cell imaging, real-time nuclear labeling and dynamic observation of live cells, cell cycle analysis, DNA labeling of live cells, research on chromatin ultrastructure, etc.
Product Features
1. No membrane lysis/washing required: Direct staining of live cells without membrane lysis, and no washing after staining;
2. Ultra-rapid staining: Fast staining in as little as 5 minutes at room temperature, greatly shortening experimental time;
3. Easy to use: Supplied as a ready-to-handle 5 mM liquid formulation, ready for use after dilution as needed;
4. Ideal for multicolor combination: Broad excitation and far-red emission bands, suitable for multicolor fluorescent labeling combinations;
5. Cell imaging compatibility: As a membrane-permeable nuclear dye, DRAQ5 can be used for live cell imaging and nucleic acid content quantification.
Product Parameters
1. Ex/Em: 601/699 nm;
2. CAS No.: 252903-95-0;
3. Molecular Formula: C₂₂H₂₈N₄O₄;
4. Molecular Weight: 412.48;
Spectrum:
Product Components
| Component | 40 Tests | 100 Tests | 400 Tests | | Ready-to-use DRAQ5 Live Cell DNA Dye (5 mM in Water) | 20 μL | 50 μL | 200 μL |
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Note: Specifications are calculated based on 5×10⁴ cells/well with a working staining solution concentration of 20 μM.
Precautions
1. DRAQ5 is a far-red fluorescent dye that is invisible to the naked eye and requires observation with a CCD camera or laser confocal microscope.
2. Due to the broad excitation and emission wavelength ranges of DRAQ5, it is not recommended to use DRAQ5 in combination with other far-red fluorescent dyes excitable by 488 nm or 633 nm laser.
3. Fluorescent dyes are subject to quenching; please protect from light as much as possible to slow down quenching.
4. Briefly centrifuge the product before use before performing subsequent operations.
5. This product is for research use only and shall not be stored in ordinary residential premises.
6. For your safety and health, comply with the general laboratory safety regulations of your institution.
Instructions for Use
I. Pre-Experiment Preparation
1. Reagent Preparation:
(1) Take the reagent out of storage conditions (e.g., 4 °C) and equilibrate to room temperature (15-25 °C).
(2) Prepare cell culture medium and buffer (e.g., azide-free PBS).
2. Instrument Preparation:
Fluorescence microscope: Equipped with a 633 nm or 647 nm light source.
II. Operational Procedures
Protocol 1: Fluorescence Microscopy Detection
Cell Preparation: Prepare azide-free PBS buffer or specific medium for specific cells. Resuspend cells with PBS or medium and control the cell density ≤4×10⁵ cells/mL. Roughly estimate the cell number for adherent cells or tissues.
2. Working Staining Solution Preparation: Dilute DRAQ5 fluorescent staining solution according to Table 1. DRAQ5 staining solution can be directly added to the surface of adherent cells or tissue sections, or directly into fresh culture medium.
Notes: (1) The optimal staining concentration of DRAQ5 may vary for different cell types; it is recommended to perform a pre-experiment for cell staining initially and set up a gradient of different DRAQ5 staining concentrations to determine the optimal one.
(2) For experiments requiring continuous monitoring (e.g., EGFP-labeled protein translocation assays), DRAQ5 staining solution should be added at a concentration of 1 μM before the addition of any agonists/antagonists (usually 0.5-3 h in advance).
Table 1. Cell Number and Corresponding DRAQ5 Volume & Final Concentration
Cell Sample Preparation
| Cell Sample Preparation
| Volume and Final Concentration of DRAQ5 Added
| Volume and Final Concentration of DRAQ5 Added
| Volume and Final Concentration of DRAQ5 Added
| | Cell Number | Volume of PBS or Medium | 5 μM | 10 μM | 20 μM | | 1×10⁶ | 2500 μL | 2.5 μL | 5 μL | 10 μL | | 4×10⁵ | 1000 μL | 1 μL | 2 μL | 4 μL | | 2×10⁵ | 500 μL | 0.5 μL | 1 μL | 2 μL | | 1×10⁵ | 250 μL | 0.25 μL | 0.5 μL | 1 μL | | 5×10⁴ | 125 μL | 0.13 μL | 0.25 μL | 0.5 μL |
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3. Cell Staining
Mix gently and incubate at room temperature for 5-30 minutes. No washing step is required after incubation.
4. Detection:
(1) Flow cytometry detection: When the dye is excited at 488 nm, detection can be performed using a 685LP dichroic mirror and a 710/50 channel; when excited at 633 nm, a 660/20 channel is applicable. For cell cycle/DNA analysis applications, it is recommended to use longer-wavelength filters (e.g., a 735LP dichroic mirror and a 780/60 channel) to optimize the CV values of G1 and G2/M peaks.
(2) Fluorescence microscopy detection: Excitation with a 633 nm or 647 nm light source is recommended.
III. Result Interpretation
Qualitative Analysis (Microscopy):
Figure 1. Fluorescence micrograph of live cell DNA staining with DRAQ5 dye
1. Normal nuclear staining: The nuclear region of live cells shows uniform red fluorescence with a clear nuclear boundary;
2. Background interference: Strong extracellular or background fluorescence indicates insufficient washing or excessively high dye concentration.
Note: The image is cited from the reference DNA labeling in living cells (doi: 10.1002/cyto.a.20172).
Frequently Asked Questions (FAQs)
1. Q: Does the DRAQ5 stock solution contain DMSO?
A: This product is an aqueous solution and contains no DMSO.
2. Q: I am performing 2-3 h live cell imaging of mouse cells with DRAQ5. How long can I image DRAQ5-stained cells before cytotoxicity occurs and causes cell death?
A: The instructions state that "DRAQ5 staining is accelerated at 37 °C, and the incubation time (5-30 min) may be shortened", but this should be determined separately by titration for each cell type. DRAQ5 can be added to the assay medium at a concentration of 1 μM for a detection duration of 0.5-3 h (typically) before the addition of any agonists/antagonists. DRAQ5 staining occurs very rapidly, so it is recommended to add DRAQ5 to live cells immediately before analysis. The incubation time with cells should not exceed 3 h, but this may vary by cell line.
3. Q: When staining the nuclei of epithelial cancer cells with DRAQ5, I found that the fluorescence becomes increasingly intense over time. This effect is more pronounced at higher laser power and with heating. What could be the cause? Does more dye bind to nuclear DNA over time? Does it only fluoresce when it reacts with nuclear DNA? Is the behavior of the dye during apoptosis known? Can the dye bind to nuclear DNA just as well when the dye concentration is low?
A: It is confirmed that DRAQ5 staining is enhanced at higher temperatures, so the incubation period may need to be adjusted. The longer the incubation period, the more signal we expect, until DNA saturation is reached. If DRAQ5 is subsequently washed away, we expect the staining to decrease slowly over time.DRAQ5 emits almost no fluorescence unless it binds to double-stranded DNA (dsDNA); it only produces a strong fluorescent signal when intercalated into dsDNA. DRAQ5 staining is not affected during apoptosis because the affinity of DRAQ5 for dsDNA is unchanged in live or dead cells. As the amount of dsDNA changes during apoptosis, the intensity of the DRAQ5 signal also changes in response to the variation of dsDNA.
4. Q: I am conducting an 18 h cell imaging experiment. When I replace the DRAQ5-containing medium with DRAQ5-free medium after 12 h, the staining intensity decreases.
① Is it necessary to keep DRAQ5 in the medium at all times, even if cytotoxic effects may occur?
② How long will the staining persist if DRAQ5 is removed from the medium?
A: ① We expect cell staining to decrease after medium replacement. Although DRAQ5 has a high affinity for dsDNA, an equilibrium exists, and the removal of low residual concentrations in the medium disrupts this equilibrium.
② We believe the staining may persist for 1-2 h after removal, but this may depend on the concentration the cells were previously exposed to. If there is any evidence of toxicity, one would want to maintain a lower concentration (possibly 0.5-1 μM). Certainly, whether DRAQ5 exerts cytotoxic effects depends entirely on the cell line and its state. Based on literature reports, it can be reasonably predicted that non-proliferating cells are less sensitive to any cell-permeable DNA intercalating probe. For example, it is more likely to be suitable for terminally differentiated cell types such as macrophages or human umbilical vein endothelial cells, where it and the closely related CyTRAKOrange have been demonstrated in long-term experiments. If the cell line used shows no toxic effects during use, then the 18 h experiment can be performed continuously in the presence of DRAQ5.
5. Q: When using DRAQ5 and DRAQ7, what are the differences between them and how to choose?
A: Choose DRAQ5 for DNA analysis of live cells, long-term monitoring of cell status, or deep tissue imaging; choose DRAQ7 for assessing cell viability, detecting apoptosis, or distinguishing live/dead cell populations in samples. The differences between the two are as follows:
| Comparison Index | DRAQ5 | DRAQ7 | | Cell Membrane Permeability | Permeable | Impermeable | | Staining Target | Nuclei of all live and fixed cells | Nuclei of only dead, apoptotic or permeabilized cells | | Binding Mode | Stoichiometric binding to DNA; fluorescence intensity reflects DNA content | Specific binding to dsDNA with rapid staining | | Applicable Scenarios | Live cell DNA content analysis, cell cycle research, long-term live cell imaging experiments, deep tissue/tumor tissue imaging | Cell viability assessment, apoptosis detection, in vitro toxicity testing of drugs/compounds, flow cytometry experiments for distinguishing live/dead cells |
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ZJ26F0636939
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