· Evaluation of single cells to detect heterogenous populations
评价检测单个细胞异质种群
· Whole blood assay: No isolation procedures and optimal culture medium
全血检测:没有隔离程序和培养基
· Physiological stimulans for phagocytes: Bacteria and fMLP
吞噬细胞生理:细菌和fMLP
· Dose response: Low and high stimulant
剂量反应:低和高的
· No electrostatic artifacts in polystyrol tubes compared to latex beads
没有静电工件比较乳胶珠子聚苯乙烯管内
· Standardized test procedure
标准化测试程序
· Exclusion of aggregation artifacts by DNA staining
通过DNA染色排除聚合工件
· Compatible with whole blood of mice and rats
兼容的小鼠和大鼠全血
· Fast assay: Whole assay time is 1.5 hours
快速测定:整个试验时间是1.5小时
100 analyses. Clinical Diagnostic for the Quantitative Analysis of Leukocyte Oxidative Burst in Human Whole Blood. Evaluation by flow cytometry.
100次分析。临床诊断人体全血白细胞氧化破裂的定量检测。通过流式细胞术进行评估。
Clinical Diagnostic for the Quantitative Analysis of Leukocyte Oxidative Burst in Human Whole Bloods
GLYCOTOPE公司PHAGOBURST白细胞氧化突发定量分析10-0200
SUMMARY and EXPLANATION
BURSTTEST (PHAGOBURST) allows the quantitative determination of leukocyte oxidative burst in heparinized whole blood. It contains unlabeled opsonized bacteria (E.coli), phorbol 12-myristate 13-acetate (PMA) and the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP) as stimulants, Dihydrorhodamine (DHR) 123 as a fluorogenic substrate and necessary reagents. It determines the percentage of phagocytic cells which produce reactive oxidants (conversion of DHR 123 to R 123) and their enzymatic activity (amount of R 123 per cell).
The evaluation of these bioactivities should be performed by flow cytometry.
BURSTTEST(PHAGOBURST)允许定量测定白细胞氧化闯入肝素化全血。 它包含未标记的促进调理作用的细菌(大肠杆菌),佛波醇12十四烷酸乙酸13(PMA)和趋化作用的多肽n甲酰遇见亮氨酸板式换热器(fMLP)作为xing奋剂,二氢若丹明(DHR)123作为一个荧光衬底和必要的试剂。它决定了吞噬细胞产生活性氧化剂的比例(转换DHR 123 到 R 1233)及其酶活性(每个细胞R 123的数量)。
这些生物活性的测定应该有流式细胞术完成。
APPLICATIONS
The diagnostic kit is intended to investigate the altered oxidative burst activity found in various disorders and to evaluate the effects of drugs.
Reduced or missing burst activity is observed in inborne defects like the chronic granulomatous disease (CGD). CGD is a heterogenous group of inherited disorders that usually manifests itself during the first two years of life (3, 4). The disease is characterized clinically by repeated and life-threatening infections caused by bacterial and fungal organisms. These infections typically consist of pneumonia, lymphadenitis, or abscesses that involve lymph nodes, lungs, and liver. The NADPH oxidase is the enzyme system responsible for producing superoxide anion, which is quickly converted to hydrogen peroxide and hydroxyl radicals. Abnormalities in the constituent peptides of the NADPH oxidase enzyme system lead to the dysfunctions characteristic of CGD. Neutrophils from CGD patients fail to produce a significant oxidative burst following stimulation. Different forms of CGD are described (classical X-linked CGD and autosomal recessive patterns). BURSTTEST (PHAGOBURST?) is a rapid and sensitive method for the diagnosis of CGD and for the detection of X-linked carriers.
The oxidative burst of granulocytes is impaired in transplant patients and patients with AIDS (6). The spontaneous and fMLP-induced neutrophil respiratory burst was shown to be increased in neonates with laboratory signs of infection (7). Various immunomodulators (e.g., cytokines (GM-CSF, G-CSF, TNF) or drugs) seem to have effects on the oxidative burst. By using fMLP as a low stimulant one can investigate additive or priming effects (8) of test substances.
The diagnostic kit is also applicable on blood of mice, rats, rabbits, dogs, cattle and other species.
TEST PRINCIPLES Phagocytosis by polymorphonuclear neutrophils and monocytes constitutes an essential arm of host defense against bacterial or fungal infections. The phagocytic process can be separated into several major stages: chemotaxis (migration of phagocytes to inflammatory sites), attachment of particles to the cell surface of phagocytes, ingestion (phagocytosis) and intracellular killing by oxygen-dependent (oxidative burst) and oxygen-independent mechanisms (1, 2).
BURSTTEST (PHAGOBURST?) allows the quantitative determination of leukocyte oxidative burst. The BURSTEST kit contains unlabelled opsonized E.coli bacteria as particulate stimulus, the protein kinase C ligand phorbol 12-myristate 13-acetate (PMA) as high stimulus and the chemotactic peptide N-formyl-MetLeuPhe (fMLP) as low physiological stimulus, Dihydrorhodamine (DHR) 123 as a fluorogenic substrate (5) and necessary reagents. Heparinized whole blood is incubated with the various stimuli at 37°C, a sample without stimulus serves as negative background control. Upon stimulation, granulocytes and monocytes produce reactive oxygen metabolites (superoxide anion, hydrogen peroxide, hypochlorous acid) which destroy bacteria inside the phagosome. Formation of the reactive oxidants during the oxidative burst can be monitored by the addition and oxidation of DHR 123. The reaction is stopped by addition of LYSING SOLUTION, which removes erythrocytes and results in a partial fixation of leukocytes. After one washing step with WASHING SOLUTION, DNA STAINING SOLUTION is added to exclude aggregation artifacts of bacteria or cells. The percentage of cells having produced reactive oxygen radicals are then analyzed as well as their mean fluorescence intensity (enzymatic activity)
注意:使用手动QIAamp DNA Blood Kit和QIAGEN Blood & Cell Culture DNA Kit的用户应该用7 ml蒸馏水重悬每个瓶中的QIAGEN Protease。
注意:使用QIAamp DNA Blood BioRobot 9604 Kit的用户应该用10 ml蒸馏水重悬每个瓶中的QIAGEN Protease。
注意:QIAGEN蛋白酶不可与DNeasy Tissue、DNeasy 96 Tissue及QIAamp DNA Mini Kit中的ATL缓冲液同时使用。在SDS浓度大于0.5%、肌氨酰浓度大于1%、或有高浓度的其他去污剂时,EDTA的浓度必须小于8 mM,这样才能在延长的孵育时间内达到an quan活性。
qiagen 73404 RNeasy Plus Universal Tissue Mini Kit
产品型号:
简单描述
qiagen 73404 RNeasy Plus Universal Tissue Mini Kit从各种类型组织中纯化总RNA,配有gDNA Eliminator Solution适用于所有组织溶液类型整合RNeasy和QIAzol技术,节省时间不使用酶,快速去除基因组DNARNA纯度高,适用于下游应用
详细介绍
qiagen 73404 RNeasy Plus Universal Tissue Mini Kit
qiagen 73404 RNeasy Plus Universal Tissue Mini Kit
从各种类型组织中纯化总RNA,配有gDNA Eliminator Solution
适用于所有组织溶液类型
整合RNeasy和QIAzol技术,节省时间
不使用酶,快速去除基因组DNA
RNA纯度高,适用于下游应用
RNeasy Plus Universal Kits可快速、方便的纯化RNA,同时有效地去除基因组DNA。优化的实验方案可从各种组织类型,包括难裂解组织中纯化高品质RNA。该试剂盒含有QIAzol Lysis Reagent和RNeasy离心柱,前者用于裂解脂肪组织和其他组织类型,后者用于纯化高品质RNA。该试剂盒有两种规格:RNeasy Plus Universal Mini Kit可从至多50 mg组织中纯化RNA,RNeasy Plus Universal Midi Kit可从至多250 mg组织中纯化RNA。RNeasy Plus Universal Mini Kit可在QIAcube全自动核酸纯化仪上自动化进行纯化。
订购信息:
Cat No./ID: 73404
RNeasy Plus Universal Mini Kit (50)
Log in
For 50 RNA minipreps: RNeasy Mini Spin Columns, gDNA Eliminator Solution, Collection Tubes, RNase-Free Water and Buffers
Cat No./ID: 73442
RNeasy Plus Universal Midi Kit (10)
Log in
For 10 RNA midipreps: RNeasy Midi Spin Columns, gDNA Eliminator Solution, Collection Tubes, RNase-Free Water and Buffers
凯杰试剂盒REPLI-g Single Cell Kit (24)应用 仪器全基因组测序二代测序平台†外显子组测序SNP基因分型芯片芯片平台†Array CGHqPCR/PCR技术Real-time PCR/PCR仪†Sanger测序毛细管测序仪†焦磷酸测序PyroMark (QIAGEN)
详细介绍
凯杰试剂盒REPLI-g Single Cell Kit (24)
Performance
覆盖基因组所有位点,十分适用于二代测序和其他下游应用
REPLI-g Single Cell Kit扩增的DNA平均长度大于10 kb,并且覆盖所有基因组位点。该产品已经过验证,适用于二代测序、基于芯片技术的比较基因组杂交(aCGH)和real-time PCR应用等多种下游分析。使用该产品无需单独进行PCR扩增,减少了手动操作,获得的产物长度PCR方法更长(参见”Next-generation sequencing using REPLI-g amplified DNA requires less hands-on time and generates more sequence information than PCR-based methods”)。用扩增产物进行二代测序,获得高品质结果。这表明该试剂盒的位点覆盖率大,错配率低,其扩增的DNA(甚至从单一细菌细胞中扩增的DNA)能够与纯化获得的基因组DNA相媲美(参见”Comparable NGS results”)。另有研究对人类常染色体和X染色体上的多个标记物进行了分析,三个独立的实验均显示:基因组的所有位点被成功扩增,没有遗漏任何一个位点(参见”Complete genome coverage”)。
REPLI-g Single Cell Kit的表现优于其他供应商的试剂盒
其他供应商的试剂盒基于PCR技术进行全基因组扩增,获得的产物片段较短,带有PCR引物序列,可能影响下游应用。基于PCR的全基因组扩增容易出现错误,会导致碱基对突变、STR的减少或增多,使用低保真Taq DNA聚合酶会导致位点缺失。相反,REPLI-g Single Cell Kit能够进行高度均一的全基因组扩增,位点偏差小。对四个试剂盒进行了检测,其中两个利用多重置换扩增技术(包括REPLI-g Single Cell Kit),另外两个利用PCR技术。采用单一细胞扩增实验方案对所有试剂盒进行序列位点检测。除REPLI-g Single Cell Kit外的其他供应商的试剂盒都出现了位点的遗漏(参见”Unbiased DNA amplification from a single cell”)。
凯杰试剂盒REPLI-g Single Cell Kit (24)
Principle
REPLI-g Single Cell Kit含有REPLI-g sc Polymerase,这是一种优化的新型高保真Phi 29聚合酶。该试剂盒采用多重置换扩增技术扩增复杂的基因组DNA,温和的碱孵育能够避免DNA片段化,促进对所有位点的无偏差扩增。该试剂盒十分适用于分离的肿瘤细胞或细菌细胞等单一细胞,能够扩增获得高产量DNA(参见表1)。此外,该试剂盒还配有专用于鲜血或干血、新鲜或冷冻组织的实验方案,可用于多种研究样本。常规DNA产量可达40 µg,对模板的起始量要求低,因此进行后续遗传分析时无需检测DNA浓度。REPLI-g Single Cell Kit的扩增产物平均长度超过10 kb,且覆盖所有位点,适合于多种应用,包括二代测序、基于芯片技术的比较基因组杂交、焦磷酸测序和real-time PCR分析(参见表2)。
表2. 下游应用和仪器
应用
仪器
全基因组测序
二代测序平台†
外显子组测序
SNP基因分型芯片
芯片平台†
Array CGH
qPCR/PCR技术
Real-time PCR/PCR仪†
Sanger测序
毛细管测序仪†
焦磷酸测序
PyroMark (QIAGEN)
† 多家供应商均有销售
扩增原理
REPLI-g Single Cell Kit采用等温基因组扩增技术,即多重置换扩增(MDA)。六聚体与变性DNA随机结合,然后在等温条件下,在优化的Phi 29聚合酶作用下,发生链置换合成反应。每个置换后的单链作为模板,与引物结合,可扩增获得高产量DNA(参见”Multiple Displacement Amplification (MDA) technology”)。Phi 29聚合酶为噬菌体衍生酶,具有3'→5'引物外切酶活性(校正活性),其保真性是Taq DNA聚合酶的1000倍。Phi 29聚合酶在优化的REPLI-g Single Cell缓冲液体系中,能够轻松的打开发卡结构等二级结构,因此可促进扩增时聚合酶正常工作。可扩增获得长达100 kb的DNA片段(参见”Unbiased amplification with Phi 29 polymerase”)。
细胞裂解和DNA的碱变性
基因组DNA在用于酶促扩增反应前必须经过变性,而这一变性过程通常使用高温或高pH值(强碱)孵育。REPLI-g Single Cell Kit采用温和的碱孵育,在细胞裂解、DNA变性的同时,确保DNA片段化程度小,不产生碱基突变。因此扩增获得的DNA完整度高,产物长度长,覆盖位点多(参见”Effect of heat and alkaline denaturation on loci representation”)。
高效去除可检测到的DNA污染
REPLI-g Single Cell Kit中的所有组分都经过了dute的去污染流程,避免扩增产物被污染。在标准化流程中,对缓冲液和试剂进行紫外线照射,确保其不会污染DNA(参见”Innovative decontamination procedure”)。紫外线照射后,对试剂盒的所有组分进行严格的质量控制,确保其性能。
Procedure
简单的单管操作
REPLI-g Single Cell Kit可对单一细胞或有限的样本进行简便、可靠、准确的全基因组扩增。反应体系构建十分便利、可靠,仅需15分钟左右的手动操作(参见”REPLI-g Single Cell Kit procedure”)。专用的缓冲液和试剂可对单一细胞、有限的组织材料、或纯化的DNA进行扩增,产量高、位点覆盖率大(参见表3)。REPLI-g技术扩增获得的DNA可在–20°C长期储存(参见”Consistent long-term stability”)。请参见表4,了解REPLI-g产品线的更多信息。
invitrogen MPK1096 Neon Transfection System 内容及储存• 3 × 1 ml Resuspension Buffer R• 3 × 1 ml Resuspension Buffer T• 2 × 150 ml Electrolytic Buffer E• 96 × 10 µl Neon® tips• 20 Neon® ele
详细介绍
invitrogen MPK1096 Neon Transfection System 10 µL Kit
描述
The Neon® Transfection System 10 µL Kit is designed specifically for use with the Neon® Transfection System. Use this kit for transfection volumes of 10 µL, containing 5 × 104–2 × 105adherent cells or 1 × 105–5 × 105 suspension cells. Cells that have been transfected using the included 10 µL Neon® Tips are ready to be washed and plated into a multi-well culture dish
invitrogen MPK1096 Neon Transfection System 10 µL Kit
规格
Cell Type:
Established Cell Lines, Hard-to-Transfect Cells, Primary Cells, Stem Cells