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彗星实验（Comet Assay），又称单细胞凝胶电泳实验（SCGE），是一种在单细胞水平上检测 DNA 损伤程度的方法。
当细胞 DNA 因化学或物理因素（如药物、辐射、重金属等）受损发生单链或双链断裂后，将细胞包埋于低熔点琼脂糖中并滴加在载玻片上。在裂解液的作用下，细胞膜、核膜及其他生物膜被破坏，细胞内的 RNA、蛋白质及其他成分进入凝胶并扩散到裂解液中，仅核 DNA 附着在剩余的核骨架上。在电场作用下，完整的大分子 DNA 会保留在原位，断裂的小分子 DNA 片段会向阳极迁移，形成头部（细胞核区域）和尾部（迁移的DNA 片段）构成的彗星状图像，用 PI 染色后可在荧光显微镜下观察（若细胞未受损伤，核 DNA 呈现圆形的荧光团，无拖尾或仅有轻微拖尾现象）。根据电泳缓冲液的 pH 值，可分为中性彗星实验（pH ≈ 8.4）和碱性彗星实验（pH＞13）。中性彗星实验主要用于检测 DNA 双链断裂（DSBs），碱性彗星实验具有更高的灵敏度，可用于检测 DNA 单链断裂（SSBs）、双链断裂以及碱性不稳定位点（ALS）。
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自备仪器和试剂
1.荧光显微镜（PI: Ex/Em=535 nm/617 nm）
2.磷酸盐缓冲液（PBS，不含 Mg²⁺和 Ca²⁺，pH7.4）、去离子水、1×TAE 或 TBE 电泳液（如有需求）
3.水浴锅、离心机、水平电泳槽、可调节式移液枪及枪头
注意事项：本产品仅供科学研究使用，不适用于临床诊断。
实验流程
1 试剂准备
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2 样本制备
1. 在进行实验前，4℃预冷（至少 30 min彗星实验裂解液和 1× 工作液碱性电泳缓冲液（如有中性电泳需求，预冷 1×TAE 或 TBE 电泳液）。
2. 将低熔点琼脂糖(LMA)瓶在90-95℃的水浴中加热20 min至LMA 完全液化，转移到 37℃的水浴中平衡 20 min。
3. 彗星实验载玻片提前放置在 37℃环境中预热 10 min。
4. 准备细胞样本，如下所示：
(1) 悬浮细胞：800 g 离心细胞 5 min，去掉上清液。用 PBS 清洗 1 次，离心去掉上清液。用 PBS 重悬细胞并计数，收集1-5 × 10⁵ 个细胞重悬于 1 mL PBS 中。
(2) 贴壁细胞：用细胞刮轻轻地从培养皿中取出细胞（或用胰蛋白酶消化，注意不要过度消化），将细胞悬液转移到离心管中，800 g 离心细胞 5 min，去掉上清液。用 PBS 清洗 1 次，离心去掉上清液。用 PBS 重悬细胞并计数，收集 1-5× 10⁵ 个细胞重悬于 1 mL PBS 中。
5. 将细胞样品与 37℃ LMA 以 1:10 的体积比混合，立即取 50 μL 混合液加入到水平放置的 37℃ 彗星实验载玻片的圆形样本孔中（将枪头抵住圆孔边缘，匀速推液同时由外向内缓慢转圈移动，使混合液均匀铺展）。
6. 保持彗星实验载玻片水平，4℃避光凝固 LMA 15 min。
7. 将彗星实验载玻片转移到干净容器中，沿容器壁缓慢加入预冷的彗星实验裂解液（液面高于载玻片），4℃避光裂解30-60 min。
注意：
(1) 正式实验前，需做预实验，确定最佳铺胶的细胞数量和裂解时间。
(2) 处理多个样品时，可将细胞样品与 LMA 的混合液保持在 37℃水浴锅中以避免凝胶化。
(3) 为避免紫外线对细胞样品造成损害，请在低/暗光条件下进行测定。
(4) 对于难以通过常规裂解充分释放 DNA 的特殊样本，可在裂解后用 DTT（二硫苏糖醇）、LIS（3,5-二碘水杨酸锂）等试剂 4℃继续处理。
3 实验步骤
电泳实验：根据您的实验目的选择合适的电泳缓冲液。
1. 中性电泳：
(1) 吸出彗星实验裂解液，沿容器壁缓慢加入预冷的 1×TAE 或 TBE 电泳液（液面高于载玻片），4℃避光浸泡 15 min。
(2) 小心地将玻片转移到水平电泳槽的凝胶平台上，加入适量预冷的 1×TAE 或 TBE 电泳液到电泳槽，直到完全覆盖住玻片（液面不得高于载玻片 0.5 cm）。
(3) 按照 1 V/cm 设置电压（例如，电泳槽正负两极的电极丝之间相距 25 cm，则施加 25 V 电压），电泳 30-45 min（电压、电流可通过改变电泳液面高低来加以调节）。
(4) 小心地将玻片从电泳槽水平转移到干净容器中，加入 50 μL/孔 PI 染料，室温避光染色 10 min。
(5) 沿容器壁缓慢加入去离子水（液面高于载玻片），清洗多余的 PI 染料，吸出去离子水。
(6) 小心取出玻片，使用荧光显微镜观察并拍照。
2. 碱性电泳：
(1) 吸出彗星实验裂解液，沿容器壁缓慢加入预冷的 1×碱性电泳缓冲液（液面高于载玻片），4℃避光解旋30 min。
(2) 小心地将玻片转移到水平电泳槽的凝胶平台上，加入适量预冷的 1×碱性电泳缓冲液到电泳槽，直到完全覆盖住玻片（液面不得高于载玻片 0.5 cm）。
(3) 按照 1 V/cm 设置电压（例如，电泳槽正负两极的电极丝之间相距 25 cm，则施加 25 V 电压），电泳 15-30 min（电压、电流可通过改变电泳液面高低来加以调节）。
(4) 小心地将玻片从电泳槽水平转移到干净容器中，沿容器壁缓慢加入去离子水（液面高于载玻片），浸洗 5 min，重复2 次。
(5) 吸出去离子水，加入 50 μL/孔 PI 染料，室温避光染色 10 min。
(6) 沿容器壁缓慢加入去离子水（液面高于载玻片），清洗多余的 PI 染料，吸出去离子水。
(7) 小心取出玻片，使用荧光显微镜观察并拍照。
注意：如无法及时拍照，将玻片置于4℃湿盒中，可短暂保存 4 h。
4 结果计算
(1) 定性分析：DNA 损伤程度按彗星尾部 DNA 百分含量分为 5 级：
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(2) 通过图像分析软件，如 OpenComet、CASPlab 和 Comet Assay IV（Perceptive Instruments）等，可对彗星图像进行定性、定量或半定量评估。常用指标包括尾长（彗星头部边缘至尾部最远端的距离）、尾部 DNA 百分含量（迁移至尾部的 DNA 荧光强度占整个彗星总荧光强度的百分比）、尾矩（尾长与尾部 DNA 百分含量的乘积）、Olive 尾矩（彗星头部中心至尾部中心距离与尾部 DNA 百分含量的乘积）、彗星率（彗星细胞数量去掉 0 级细胞后占总细胞数量的百分比）、总评分（各损伤等级细胞占比与其对应级别号的乘积之和）等。每个样品至少应分析 50 个细胞。
 图1.彗星结果分析示意图 |
5 结果示例
图 2. Hela 细胞未经处理（左）或处理（右，UV 照射 15 min）进行碱性彗星实验（21 V，电泳 15 min），染色后检测的效果图。 数据仅供参考。 |
6 FAQ
(1) 组织样本如何处理？
将组织参考文献方法例如使用胰酶、胶原酶、EDTA 等消化处理制备成单细胞悬液，再按照悬浮细胞进行处理。
(2) 染色效果不好如何优化？
可以延长裂解时间，甚至可过夜孵育（此处可作为本实验的一个长时间暂停点，不会影响实验结果）。
The Comet Assay, also known as Single Cell Gel Electrophoresis (SCGE), is a method for detecting the degree of DNA damage at the single-cell level.
When cellular DNA suffers single-strand or double-strand breaks induced by chemical or physical stimuli (e.g., drugs, radiation, heavy metals), cells are embedded in low melting point agarose and loaded onto slides. Lysis buffer disrupts cell membranes, nuclear membranes and other biomembranes; intracellular RNA, proteins and other components diffuse into the lysis solution, leaving only nuclear DNA anchored to the residual nuclear scaffold. Under an electric field, intact high-molecular-weight DNA remains in situ, while fragmented small DNA fragments migrate toward the anode to form a comet-shaped image consisting of a head (nuclear region) and a tail (migrated DNA fragments). After staining with Propidium Iodide (PI), samples can be visualized under a fluorescence microscope. Undamaged cells exhibit a round fluorescent nucleus with no or minimal tailing.
Based on the pH of electrophoresis buffer, two assay formats are available: neutral comet assay (pH ≈ 8.4) and alkaline comet assay (pH>13). The neutral comet assay specifically detects DNA double-strand breaks (DSBs). The alkaline comet assay delivers higher sensitivity, capable of detecting DNA single-strand breaks (SSBs), double-strand breaks and alkali-labile sites (ALS).
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Required Instruments and Reagents
Fluorescence microscope (PI: Ex/Em = 535 nm/617 nm)
Phosphate Buffered Saline (PBS, Mg²⁺/Ca²⁺ free, pH 7.4), deionized water, 1×TAE or TBE electrophoresis buffer (if needed)
Water bath, centrifuge, horizontal electrophoresis tank, adjustable pipettes and pipette tips
Notes: For research use only. Not intended for clinical diagnostic applications.
Assay Protocol
1 Reagent Preparation
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2 Sample Preparation
Pre-chill Comet Lysis Buffer and 1×Alkaline Electrophoresis Solution at 4 ℃ for a minimum of 30 min before use; pre-chill 1×TAE/TBE buffer if performing neutral electrophoresis.
Heat Low Melting Agarose (LMA) vial in a 90–95 ℃ water bath for 20 min until fully liquefied, then equilibrate in a 37 ℃ water bath for 20 min to maintain liquid state.
Pre-warm Comet Slides at 37 ℃ for 10 min in advance.
Prepare cell suspensions as follows:
(1) Suspension cells: Centrifuge cells at 800 g for 5 min, discard supernatant. Wash once with PBS, re-centrifuge and remove supernatant. Resuspend cells in PBS and count; collect 1–5×10⁵ cells and resuspend in 1 mL PBS.
(2) Adherent cells: Gently scrape cells from culture dishes with a cell scraper (or digest with trypsin, avoid over-digestion). Transfer cell suspension to centrifuge tubes, centrifuge at 800 g for 5 min and discard supernatant. Wash once with PBS, re-centrifuge and remove supernatant. Resuspend cells in PBS and count; collect 1–5×10⁵ cells and resuspend in 1 mL PBS.
Mix cell suspension with pre-equilibrated 37 ℃ LMA at a volume ratio of 1:10. Immediately pipette 50 μL mixture into the circular well of pre-warmed (37 ℃) horizontal Comet Slide. Touch pipette tip to the edge of the well, dispense liquid evenly while circling outward to inward for uniform spreading.
Keep Comet Slide horizontal and solidify LMA at 4 ℃ in the dark for 15 min.
Transfer Comet Slide to a clean container, slowly add pre-chilled Comet Lysis Buffer along the wall to fully submerge slides, then lyse at 4 ℃ protected from light for 30–60 min.
Notes:
(1) Perform preliminary experiments to optimize cell seeding density and lysis duration.
(2) For batch sample processing, keep cell-LMA mixture in a 37 ℃ water bath to prevent premature gelation.
(3) Conduct all operations under dim light to avoid UV-induced DNA damage.
(4) For hard-to-lyse special samples, further incubate with DTT (Dithiothreitol) or LIS (Lithium 3,5-Diiodosalicylate) at 4 ℃ post lysis.
3 Electrophoresis Procedure
Select appropriate electrophoresis buffer according to experimental objectives.
1.Neutral Electrophoresis
(1) Remove Comet Lysis Buffer, slowly add pre-chilled 1×TAE/TBE buffer to submerge slides, incubate at 4 ℃ in the dark for 15 min.
(2) Carefully transfer Comet Slides to the gel platform of horizontal electrophoresis tank, add pre-chilled 1×TAE/TBE buffer until slides are fully covered (liquid level shall not exceed slides by more than 0.5 cm).
(3) Set voltage at 1 V/cm (e.g., 25 V for electrodes spaced 25 cm apart), run electrophoresis for 30–45 min. Voltage and current can be adjusted by altering buffer liquid height.
(4) Horizontally transfer Comet Slides to a clean container, add 50 μL PI staining solution per well, incubate at room temperature in the dark for 10 min.
(5) Slowly add deionized water to submerge slides, rinse excess PI dye and discard water.
(6) Retrieve slides and observe & capture images under a fluorescence microscope.
2.Alkaline Electrophoresis
(1) Discard Comet Lysis Buffer, slowly add pre-chilled 1×Alkaline Electrophoresis Solution to submerge slides, unwind DNA at 4 ℃ protected from light for 30 min.
(2) Carefully transfer Comet Slides to the gel platform of horizontal electrophoresis tank, add pre-chilled 1×Alkaline Electrophoresis Solution until slides are fully covered (liquid level shall not exceed slides by more than 0.5 cm).
(3) Set voltage at 1 V/cm (e.g., 25 V for electrodes spaced 25 cm apart), run electrophoresis for 15–30 min. Voltage and current can be adjusted by altering buffer liquid height.
(4) Horizontally transfer Comet Slides to a clean container, add deionized water to submerge slides, rinse for 5 min and repeat twice.
(5) Discard deionized water, add 50 μL PI staining solution per well, incubate at room temperature in the dark for 10 min.
(6) Slowly add deionized water to submerge slides, rinse excess PI dye and discard water.
(7) Retrieve slides and observe & capture images under a fluorescence microscope.
Notes: If imaging cannot be performed immediately, store Comet Slides in a humid box at 4 ℃ for a maximum of 4 hours.
4 Data Analysis
(1) Qualitative grading: DNA damage is classified into 5 grades based on the percentage of tail DNA:
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(2) Comet images can be qualitatively, quantitatively or semi-quantitatively analyzed using software including OpenComet, CASPlab and Comet Assay IV (Perceptive Instruments). Key indicators include: Tail length: Distance from the edge of the comet head to the farthest tip of the tail, Tail DNA percentage: Ratio of fluorescent intensity of migrated tail DNA to total fluorescent intensity of the entire comet, Tail moment: Product of tail length and tail DNA percentage, Olive tail moment: Product of the distance from the center of the comet head to the center of the tail and tail DNA percentage, Comet rate: Percentage of damaged comet cells (excluding Grade 0 cells) among total counted cells. Total score: Sum of the product of the proportion of cells in each damage grade and the corresponding grade number
A minimum of 50 cells shall be analyzed per sample.
Figure 1. Schematic diagram of comet assay result analysis.
5 Representative Results
Figure 2. Representative alkaline comet assay images of untreated HeLa cells (left) vs UV-irradiated HeLa cells (15 min UV exposure, right). Electrophoresis condition: 21 V, 15 min. Data for reference only.
6 FAQ
(1) How to process tissue samples?
Digest tissue with trypsin, collagenase, EDTA or other reagents as referenced in published protocols to prepare single-cell suspension, then process following the suspension cell protocol above.
(2) How to optimize weak staining performance?
Extend lysis incubation time, even overnight; this serves as a stable stopping point without compromising assay results.
| 组分货号 | 组分名称 | 物理外观 | 15T | 储存 |
| C1526218A | 三孔彗星实验载玻片 | 固体 | 5 EA | 2-8℃. |
| C1526218B | 彗星实验裂解液 | 液体 | 75 mL×2 | 2-8℃. |
| C1526218C | 20×碱性电泳缓冲液 | 液体 | 100 mL | 2-8℃. |
| C1526218D | 低熔点琼脂糖 (LMA) | 液体 | 2 mL | 2-8℃. |
| C1526218E | 碘化丙啶(PI) | 液体 | 1 mL | 2-8℃. 避光 |
| C1526218F | 二甲基亚砜 | 液体 | 15 mL | 2-8℃. 避光 |
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