细胞核是一个功能单位，完整的保存了遗传物质，并指导RNA的合成。RNA是蛋白质及其他细胞组分合成所必须的。在大多数细胞类型中，由于核被膜与内质网的连续性以及细胞核表面与细胞骨架之间的连接等原因，细胞核是被固定于细胞中的。流式细胞术是一种快速、准确、高效的测定和分析细胞DNA含量的方法，近年来已广泛应用于植物研究中，如细胞周期分析、植物倍性鉴定、染色体分拣、细胞核DNA含量测定、生殖途径鉴定、DNA变异分析、遗传稳定性分析和体胚发生分析等。只通过物理方法即切碎叶片往往不能获取大量完整的细胞核，还需要加入一些特定成分的缓冲液，使植物细胞破损，分散细胞器，进而解离出细胞核，为后续的核DNA提取和DNA含量分析做准备。流式细胞术测定的生物细胞必须处于单细胞悬液状态，制备优质的细胞核悬液的关键在于选择合适的细胞核分离缓冲液。不同植物的组织结构和化学成分存在较大差异，使用细胞核分离缓冲液的效果不同，而且目前没有一种普遍适用的细胞核分离缓冲液，因此需要尝试不同的缓冲液，甚至改进其成分，以获得最佳的细胞核分离效果。
细胞核分离缓冲液 (Nuclear Separation Buffer，NSB)又称细胞核解离缓冲液 (Dissociation Buffer)，可以将细胞核跟细胞膜、内质网、细胞骨架等胞内成分分离开来。目前常用的解离液有Galbraith、Tris-MgCl₂、HEPES、WPB、LB01、mGb、Marie、GPB、OTTO和Marie等，各种解离液成分和浓度各不相同，不同的植物样品需要选择相适应的解离液。植物细胞核重悬缓冲液主要由20mM Hepes、蔗糖、氯化镁、DTT、甘油等组成，pH7.8±0.1。经过植物组织细胞核分离缓冲液处理后，植物细胞核重悬缓冲液用于细胞核的重悬和保存。该试剂仅用于科研领域，不适用于临床诊断或其他用途。
操作步骤 (仅供参考)：
细胞核微量提取方法：
1、取新鲜的植物样品0.1-0.5g，用蒸馏水冲洗，滤纸吸干水分，用锋利的刀片切碎，置于匀浆器中，加入2ml解离液 (自备)，匀浆1-2min。
2、用2层无菌平纹纱布过滤匀浆物，再用2ml解离液冲洗匀浆器和纱布，合并滤液。
3、室温下70r/min 离心5min，转移上清液至新离心管中，700r/min离心15min，去除上清液，保留下层细胞核沉淀。
4、核沉淀可重新悬浮于植物细胞核重悬缓冲液中，-70℃保存，用于核裂解、核DNA提取和纯化等下游实验。
注意事项：
1、样品量过少或者匀浆不充分可能会导致获得的细胞核很少，影响下游实验。
2、解离液选择不合适、样品保存不当、样品反复冻融、提取过程过分剧烈都可能导致提取的DNA断裂或降解。
3、植物细胞核重悬缓冲液含蔗糖和HEPES，易受细菌污染，建议首次溶解后分装小份冻存。
4、“不同解离液的成分及作用”和“不同解离液的成分及适用的样本”见附表。
5、试剂开封后请尽快使用，以防影响后续实验效果。
6、为了您的安全和健康，请穿实验服并戴一次性手套操作。
附表：（以下内容来源于论文资料，仅供参考）
不同解离液的成分及作用
| 成分 | 作用 | 注释 | 相关的解离液 | | 氯化镁、硫酸镁 | 稳定核染色质 | 镁离子 | G、T、H、W 、m | | 精胺、亚精胺 | 稳定核染色质 | 精胺 | L、GP、 | | 氯化钾、氯化钠 | 提供一定的离子强度，维持渗透压 | 无机盐 | H、W、L、GP、M | | 曲拉通 | 释放核，移走细胞质，分散叶绿体，减少核粘连 | 表面活性剂 | G、T、H、W、L、m、GP | | 吐温 | 释放核，移走细胞质，分散叶绿体，减少核粘连 | 表面活性剂 | O、M | | EDTA | 结合作为核酸酶辅助因子的二价阳离子，抑制核酸酶活性 | 金属螯合剂及替代物 | W、L、m、M | | 柠檬酸钠 | 结合作为核酸酶辅助因子的二价阳离子，抑制核酸酶活性 | 金属螯合剂及替代物 | G、GP、O、m、M | | 葡萄糖 | 维持核的完整性，防止核粘连 |
| M | | Tris | 维持核的完整性，防止核粘连，稳定pH | 缓冲剂 | T、W、L | | MOPS | 稳定溶液的pH | 缓冲剂 | G、m、GP | | HEPES | 稳定溶液的pH | 缓冲剂 | H、M | | PVP | 消除酚等粘性物质的影响 |
| W、m | | DTT | 保护染色质蛋白，消除酚等粘性物质的影响 | 还原剂 | H | | 2-巯基乙醇 | 保护染色质蛋白，消除酚等粘性物质的影响 | 还原剂 | L、m、M | | 焦亚硫酸钠 | 保护染色质蛋白，消除酚等粘性物质的影响 | 还原剂 | W |
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不同解离液的成分及适用的样本
| 解离液名称(简写) | 成分 | 成功测定的样本 | | Galbraith解离液(G) | 氯化镁、柠檬酸钠、MOPS、曲拉通 | 松属，番茄，杉木 | | Tris-MgCl₂解离液(T) | Tris、氯化镁、曲拉通 | 矢车菊，欧洲朴树 | | HEPES解离液(H) | HEPES、硫酸镁、氯化钾、DTT、曲拉通 | 杉木 | | WPB解离液(W) | Tris、氯化镁、EDTA、氯化钠、焦亚硫酸钠、PVP、曲拉通 | 虞美人，意大利松，欧洲李，桃，梨，夏栎，蔷薇，葡萄，垂柳 | | LB01解离液(L) | Tris、精胺、EDTA、氯化钠、氯化钾、曲拉通、巯基乙醇 | 蚕豆，山药，罗勒属，欧洲云杉，茄子，番茄 | | mGb解离液(m) | MOPS、PVP、氯化镁、EDTA、柠檬酸钠、曲拉通、巯基乙醇 | 千里光属，拟南芥 | | GPB解离液(GP) | 精胺、柠檬酸钠、氯化钾、氯化钠、曲拉通、MOPS | 胡杨，灰叶胡杨，虎榛子，豌豆，马鞍藤，葱 | | OTTO解离液(O) | 柠檬酸、吐温、磷酸氢二钠 | 沙棘属，毛茛科，杉木 | | Marie解离液(M) | 吐温、葡萄糖、氯化钾、氯化钠、EDTA、柠檬酸钠、HEPES、巯基乙醇 | 杉木 |
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The nucleus is a functional unit that fully preserves genetic material and directs RNA synthesis. RNA is essential for the synthesis of proteins and other cellular components. In most cell types, the nucleus is anchored within the cell due to the continuity between the nuclear envelope and the endoplasmic reticulum, as well as the connections between the nuclear surface and the cytoskeleton. Flow cytometry is a rapid, accurate, and efficient method for determining and analyzing cellular DNA content. In recent years, it has been widely applied in plant research, such as cell cycle analysis, plant ploidy identification, chromosome sorting, nuclear DNA content measurement, reproductive pathway identification, DNA variation analysis, genetic stability analysis, and somatic embryogenesis analysis. Physical methods alone, such as chopping leaves, often fail to yield a large number of intact nuclei. It is also necessary to add buffer solutions containing specific components to disrupt plant cells, disperse organelles, and further dissociate nuclei, laying the foundation for subsequent nuclear DNA extraction and DNA content analysis. Biological cells to be analyzed by flow cytometry must be in a single-cell suspension state. The key to preparing high-quality nuclear suspensions lies in selecting an appropriate nuclear isolation buffer. There are significant differences in tissue structure and chemical composition among different plant species, leading to varying effectiveness of nuclear isolation buffers. Currently, there is no universally applicable nuclear isolation buffer. Therefore, it is necessary to test different buffers or even modify their components to achieve the optimal nuclear isolation effect.
Nuclear Separation Buffer (NSB), also known as Dissociation Buffer, can separate the nucleus from intracellular components such as the cell membrane, endoplasmic reticulum, and cytoskeleton. Commonly used dissociation buffers at present include Galbraith, Tris-MgCl₂, HEPES, WPB, LB01, mGb, Marie, GPB, OTTO, etc. Each type of dissociation buffer varies in composition and concentration, and different plant samples require the selection of a suitable matching dissociation buffer. Plant Nucleus Resuspension Buffer is mainly composed of 20 mM HEPES, sucrose, magnesium chloride, DTT, glycerol, etc., with a pH of 7.8±0.1. After plant tissue is treated with Nuclear Isolation Buffer, Plant Nucleus Resuspension Buffer is used for the resuspension and preservation of nuclei. This reagent is for research use only and not intended for clinical diagnosis or any other purposes.
Operating Procedures (for reference only)
Micromethod for Nuclear Extraction
1. Take 0.1-0.5 g of fresh plant sample, rinse it with distilled water, blot dry with filter paper, chop it into pieces with a sharp blade, place it in a homogenizer, add 2 ml of self-prepared dissociation buffer, and homogenize for 1-2 minutes.
2. Filter the homogenate through two layers of sterile plain gauze, rinse the homogenizer and gauze with another 2 ml of dissociation buffer, and combine the filtrates.
3. Centrifuge the combined filtrate at 70 rpm for 5 minutes at room temperature. Transfer the supernatant to a new centrifuge tube, then centrifuge at 700 rpm for 15 minutes. Discard the supernatant and retain the nuclear pellet at the bottom.
4. The nuclear pellet can be resuspended in Plant Nucleus Resuspension Buffer and stored at -70℃ for downstream experiments such as nuclear lysis, nuclear DNA extraction, and purification.
Precautions
1. Insufficient sample amount or inadequate homogenization may result in a low yield of nuclei, which will affect subsequent experiments.
2. Inappropriate selection of dissociation buffer, improper sample storage, repeated freeze-thaw cycles of samples, or overly vigorous extraction processes may cause fragmentation or degradation of the extracted DNA.
3. Plant Nucleus Resuspension Buffer contains sucrose and HEPES, which are susceptible to bacterial contamination. It is recommended to aliquot the buffer into small portions and store them frozen immediately after the first dissolution.
4. See the attached table for "Components and Functions of Different Dissociation Buffers" and "Components of Different Dissociation Buffers and Their Suitable Samples".
5. Please use the reagent as soon as possible after opening to avoid affecting the results of subsequent experiments.
6. For your safety and health, wear a lab coat and disposable gloves during operation.
Attached Table (Data sourced from published papers, for reference only)
Components and Functions of Different Dissociation Buffers
| Components | Functions | Notes | Related Dissociation Buffers | | Magnesium chloride, Magnesium sulfate | Stabilize nuclear chromatin | Magnesium ions | G, T, H, W, m | | Spermine, Spermidine | Stabilize nuclear chromatin | Spermine | L, GP | | Potassium chloride, Sodium chloride | Provide a certain ionic strength and maintain osmotic pressure | Inorganic salts | H, W, L, GP, M | | Triton X-100 | Release nuclei, remove cytoplasm, disperse chloroplasts, reduce nuclear adhesion | Surfactant | G, T, H, W, L, m, GP | | Tween | Release nuclei, remove cytoplasm, disperse chloroplasts, reduce nuclear adhesion | Surfactant | O, M | | EDTA | Bind divalent cations serving as nuclease cofactors and inhibit nuclease activity | Metal chelator & substitute | W, L, m, M | | Sodium citrate | Bind divalent cations serving as nuclease cofactors and inhibit nuclease activity | Metal chelator & substitute | G, GP, O, m, M | | Glucose | Maintain nuclear integrity and prevent nuclear adhesion | - | M | | Tris | Maintain nuclear integrity, prevent nuclear adhesion, and stabilize pH | Buffer | T, W, L | | MOPS | Stabilize the pH of the solution | Buffer | G, m, GP | | HEPES | Stabilize the pH of the solution | Buffer | H, M | | PVP | Eliminate the interference of viscous substances such as phenols | - | W, m | | DTT | Protect chromatin proteins and eliminate the interference of viscous substances such as phenols | Reducing agent | H | | 2-Mercaptoethanol | Protect chromatin proteins and eliminate the interference of viscous substances such as phenols | Reducing agent | L, m, M | | Sodium metabisulfite | Protect chromatin proteins and eliminate the interference of viscous substances such as phenols | Reducing agent | W |
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Components of Different Dissociation Buffers and Their Suitable Samples
| Dissociation Buffer (Abbreviation) | Components | Successfully Tested Samples | | Galbraith (G) | Magnesium chloride, Sodium citrate, MOPS, Triton X-100 | Pinus, Solanum lycopersicum, Cunninghamia lanceolata | | Tris-MgCl₂ (T) | Tris, Magnesium chloride, Triton X-100 | Centaurea cyanus, Celtis australis | | HEPES (H) | HEPES, Magnesium sulfate, Potassium chloride, DTT, Triton X-100 | Cunninghamia lanceolata | | WPB (W) | Tris, Magnesium chloride, EDTA, Sodium chloride, Sodium metabisulfite, PVP, Triton X-100 | Papaver rhoeas, Pinus pinea, Prunus domestica, Prunus persica, Pyrus pyrifolia, Quercus robur, Rosa spp., Vitis vinifera, Salix babylonica | | LB01 (L) | Tris, Spermine, EDTA, Sodium chloride, Potassium chloride, Triton X-100, 2-Mercaptoethanol | Vicia faba, Dioscorea opposita, Ocimum spp., Picea abies, Solanum melongena, Solanum lycopersicum | | mGb (m) | MOPS, PVP, Magnesium chloride, EDTA, Sodium citrate, Triton X-100, 2-Mercaptoethanol | Senecio spp., Arabidopsis thaliana | | GPB (GP) | Spermine, Sodium citrate, Potassium chloride, Sodium chloride, Triton X-100, MOPS | Populus euphratica, Populus pruinosa, Ostryopsis davidiana, Pisum sativum, Ipomoea pes-caprae, Allium fistulosum | | OTTO (O) | Citric acid, Tween, Disodium hydrogen phosphate | Hippophae spp., Ranunculaceae, Cunninghamia lanceolata | | Marie (M) | Tween, Glucose, Potassium chloride, Sodium chloride, EDTA, Sodium citrate, HEPES, 2-Mercaptoethanol | Cunninghamia lanceolata |
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ZJ26F0332987
危险品化学品经营许可证（带存储）