- Description
- Additional Information
- Readable Documents
- Assay Principle
- Reviews
Key Benefits
- Safe – Non Radioactive Enzyme release assay.
- Versatile – Useful for measuring activity of T Cells, Primary Cells, NK, complement and other lytic agents.
- Assay can be run in serum supplemented media.
- Homogenous – One-step, no wash assay. Assay can be run in same plate as samples.
- FAST – Results in 3-5 minutes. Chromium 51 or europium release for measurement are time consuming. The inherent sensitivity of luciferase detection is enhanced by the amplification effect of enzyme turnover, which produces thousands, millions or billions of high – energy molecules for each molecule of the enzyme.
- Highly Sensitive – Can detect fewer than 500 cells/well in the presence of serum or as few as 10 cells/well in serum-free or heat-killed media.
- GAPDH: The fact that GAPDH is a natural component of cells, and does not need to be introduced into the cells in any manner, distinguishes this assay from all methods which require prelabelling of cells, transfection, transformation, or other methods of introducing proteins or other molecules into the target cells in order to generate a signal in a later step.
- Advantages for measurement of cell mediated or complement mediated cytolysis – It is usually desirable to use smaller numbers of TCells than are needed for the 51Cr – release assay, since excessive numbers of effector cells can increase the background signal. This is now possible due to the high sensitivity of aCella-Tox.
- ADCC / CMC Assays – A non radioactive alternative to 51Cr assays. Please click here for a direct comparison between the aCella-TOX and (51Cr) Chromium Release Methods
- HTS – Adaptable for High Throughput format
- Non-destructive assay allows monitoring of additional parameters.
Additional information
Kit Size | 500, 1000 |
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Includes | No Plates, 5 Lumi Plates, 5 Lumi Plates + 5 Tissue Culture Plates |
GAPDH is an important enzyme in the glycolysis and gluconeogenesis pathways. This homotetrameric enzyme catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate in the presence of cofactor and inorganic phosphate. In the aCella-TOX reaction scheme the release of GAPDH is coupled to the activity of the enzyme 3-Phosphoglyceric Phosphokinase (PGK) to produce ATP. ATP is detected via the luciferase, luciferin Bioluminescence methodology. Further, aCella-TOX is a homogeneous cytotoxicity assay; alternatively in dual mode, aCella-TOX can measure cytotoxicity and cell viability in the same plate. Culture supernatants can also be removed from the original plate and assayed in a different plate, allowing kinetics runs to be set up. The assay is non-destructive, allowing the monitoring of additional parameters such as gene expression.
Document Title |
aCella-TOX v1_3 Protocol |
aCella-TOX Datasheet |
msds.aCella-TOX |
Title | File | Link | Author(s) | Journal | Year; Edition:Pages |
Heat shock enhances the expression of cytotoxic granule proteins and augments the activities of tumor-associated antigen-specific cytotoxic T lymphocytes. | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3468674/ | Takahashi A, Torigoe T, Tamura Y, et al. | Cell Stress & Chaperones | 2012;17(6):757-763 | |
IGF-1R peptide vaccines/mimics inhibit the growth of BxPC3 and JIMT-1 cancer cells and exhibit synergistic antitumor effects with HER-1 and HER-2 peptides. | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4368154/ | Foy KC, Miller MJ, Overholser J, Donnelly SM, Nahta R, Kaumaya PT | Oncoimmunology | 2014;3(11):e956005 | |
HER-3 peptide vaccines/mimics: Combined therapy with IGF-1R, HER-2, and HER-1 peptides induces synergistic antitumor effects against breast and pancreatic cancer cells. | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4368151/ | Miller MJ, Foy KC, Overholser JP, Nahta R, Kaumaya PT | Oncoimmunology | 2014;3(11):e956012 | |
Phase I Active Immunotherapy With Combination of Two Chimeric, Human Epidermal Growth Factor Receptor 2, B-Cell Epitopes Fused to a Promiscuous T-Cell Epitope in Patients With Metastatic and/or Recurrent Solid Tumors. | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2773479/ | Kaumaya PTP, Foy KC, Garrett J, et al. | Journal of Clinical Oncology | 2009;27(31):5270-5277 | |
Identification of Cellular Proteins Required for Replication of Human Immunodeficiency Virus Type 1. | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3448097/ | Dziuba N, Ferguson MR, O"Brien WA, et al. | AIDS Research and Human Retroviruses | 2012;28(10):1329-1339 | |
Insulin-Like Growth Factor-1 Receptor Signaling Increases the Invasive Potential of Human Epidermal Growth Factor Receptor 2-Overexpressing Breast Cancer Cells via Src-Focal Adhesion Kinase and Forkhead Box Protein M1. | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4293451/ | Sanabria-Figueroa E, Donnelly SM, Foy KC, et al. | Pharmacology | 2015;87(2):150-161 | |
Combination Treatment with HER-2 and VEGF Peptide Mimics Induces Potent Anti-tumor and Anti-angiogenic Responses in Vitro and in Vivo. | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3075707/ | Foy KC, Liu Z, Phillips G, Miller M, Kaumaya PTP | The Journal of Biological Chemistry | 2011;286(15):13626-13637 | |
Resistance to Cytarabine Induces the Up-regulation of NKG2D Ligands and Enhances Natural Killer Cell Lysis of Leukemic Cells. | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2586691/ | Ogbomo H, Michaelis M, Klassert D, Doerr HW, Cinatl J. | Neoplasia (New York, NY) | 2008;10(12):1402-1410 | |
Anti-Tumor Effects of Peptide Therapeutic and Peptide Vaccine Antibody Co-targeting HER-1 and HER-2 in Esophageal Cancer (EC) and HER-1 and IGF-1R in Triple-Negative Breast Cancer (TNBC). | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4586465/ | Overholser J, Ambegaokar KH, Eze SM, et al. | Disis ML (Nora), ed. Vaccines | 2015;3(3):519-543 | |
Generation and preclinical characterization of an antibody specific for SEMA4D. | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4966508/ | Fisher TL, Reilly CA, Winter LA, et al. | mAbs | 2016;8(1):150-162 | |
A Human Anti-M2 Antibody Mediates Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Cytokine Secretion by Resting and Cytokine-Preactivated Natural Killer (NK) Cells. | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4411161/ | Simhadri VR, Dimitrova M, Mariano JL, et al. | Reeves RK, ed. PLoS ONE | 2015;10(4):e0124677 | |
Natural Cytotoxicity Receptor-Dependent Natural Killer Cytolytic activity Directed at Hepatitis C Virus (HCV) Is Associated With Liver Inflammation, African American Race, IL28B Genotype, and Response to Pegylated Interferon/Ribavirin Therapy in Chronic HCV Infection. | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3997579/ | Meng Q, Rani MRS, Sugalski JM, et al. | The Journal of Infectious Diseases | 2014;209(10):1591-1601 | |
Myxoma Virus Infection Promotes NK Lysis of Malignant Gliomas In Vitro and In Vivo. | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3677932/ | Ogbomo H, Zemp FJ, Lun X, et al. | Ulasov I, ed. PLoS ONE | 2013;8(6):e66825 | |
Targeting a Glioblastoma Cancer Stem Cell Population Defined by EGF Receptor Variant III. | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5661963/ | Emlet DR, Gupta P, Holgado-Madruga M, et al. | Cancer research | 2014;74(4):1238-1249 | |
Genetically Associated CD16+56− Natural Killer Cell Interferon (IFN)-αR Expression Regulates Signaling and Is Implicated in IFN-α-Induced Hepatitis C Virus Decline. | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3295604/ | Conry SJ, Meng Q, Hardy G, et al. | The Journal of Infectious Diseases | 2012;205(7):1131-1141 |
Reference |
Methods and compositions for coupled luminescent assays. United States Patent 6,811,990 Corey and Kinders, issued November 2, 2004. |
Corey, M. J. and Kinders, R. J. (2005) "Coupled Luminescent Methods in Drug Discovery: 3-Min Assays for Cytotoxicity and Phosphatase Activity" Drug Discovery Handbook, Ed. Shayne Cox Gad, published by John Wiley & Sons, Inc., pp. 689-731 |
Corey, M.J., et al., "A Very Sensitive Coupled Luminescent Assay for Cytoxicity and Complement-Mediated Lysis," Journal of Immunological Methods 207:43-51, 1997. |
Corey, M. J., et al., Mechanistic Studies of the Effects of Anti-factor H Antibodies on Complement-mediated Lysis,” Journal of Biological Chemistry 275: 12917-12925, 2000. |
Schafer, H., et al., "A Highly Sensitive Cytotoxicity Assay Based on the Release of Reporter Enzymes, From Stably Transfected Cell Lines," Journal of Immunological Methods 204:89-98, 1997. |
Racher, LDH Assay, in Cell and tissue culture: Laboratory procedures in biotechnology, A. Doyle and J.B. Griffiths, Eds. 1998, John Wiley & Sons: Chichester, New York, Weinheim. p. 71-5 |
Decker, T. and Lohmann-Matthes, M.L. (1988) A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J. Immunol. Meth. 115, 61-9. |
Korzeniewski, C. and Callewaert, D.M. (1983) An enzyme-release assay for natural cytotoxicity. J. Immunol. Meth.64, 313-20. |
Crouch, S.P.M., et al., "The Use of ATP Bioluminescence as a Measure of Cell Proliferation and Cytotoxicity," Journal of Immunological Methods 160:81-88, 1993. |
Henry Ogbomo, Anke Hahn, Janina Geiler, Martin Michaelis, Hans Wilhelm Doerr, Jindrich Cinatl Jr. NK sensitivity of Neuroblastoma cells determined by a highly sensitive coupled luminescent method;Biochemical and Biophysical Research Comunications 339 (2006) pp375-379. Click here to read the publication |
Part# | Reagent | Temperature |
Part # 6001 | 4X Enzyme Assay Reagent | -20C |
Part # 3008 | 1X Enzyme Assay Diluent | 2-8C |
Part # 6003 | Glyeraldehyde 3-Phosphate (G3P) | -20C |
Part # 6002 | 50X Detection Reagent | -20C |
Part # 3009 | 5.5X Detection Assay Diluent | -20C |
Part # 3035 | Lytic Agent | 2-8C |
N/A | 5 Lumi Plates (Catalog# CLATOX100-3L) | N/A |
N/A | 5 Lumi Plates + 5 Tissue Culture Plates (Catalog# CLATOX100-3P) | N/A |
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2月16日,美国专利及商标局传来重磅消息——该部门宣布,隶属于哈佛大学与麻省理工学院的Broad研究所继续保有2014年获批的CRISPR-Cas9应用专利,也让这项**性基因编辑工具的专利之争大体尘埃落定。
▲三行文字,决定了这项专利的归属(图片来源:STAT)
毫无疑问,CRISPR-Cas9基因编辑系统是本世纪最为重要的生物发现之一。2015年,《科学》将它评为年度突破;助力这项技术诞生的科学家们也先后获得了有“科学界奥斯卡”之称的“突破奖”(BreakthroughPrize),在分子生物学界影响深远的“格鲁伯遗传学奖”(GruberGeneticsPrize),以及表彰重大生物医学突破的“沃伦·阿尔珀特奖”(WarrenAlpertPrize)。
CRISPR-Cas9基因编辑系统能取得今天的成功,绝非一名科学家的功劳。2012年,JenniferDoudna教授与EmmanuelleCharpentier教授在《科学》杂志上发表文章,确认CRISPR-Cas9系统在体外实验中能“定点”对DNA进行切割。两个月后,VirginijusSiksnys教授在《PNAS》杂志上发表了类似的研究。这些论文表明CRISPR-Cas9系统作为基因编辑工具的巨大潜力。
2013年,张锋教授的研究团队在《科学》杂志上发表了一篇重磅研究:他们首次在哺乳动物内应用了CRISPR-Cas9系统,并确认它能在几周内建立起小鼠的疾病模型。此外,张锋教授的团队也首次在人体细胞内成功地用CRISPR-Cas9系统完成了基因编辑。
▲张锋教授团队率先在哺乳动物细胞中应用了CRISPR-Cas9技术(图片来源:STAT)
科学突破需要群策群力,专利申请却并非如此。2012年,加州大学伯克利分校与Broad研究所/麻省理工学院先后向美国专利及商标局递交了CRISPR应用的专利申请。2014年4月,美国专利及商标局为后者率先颁发了专利,而前者的申请至今未得到批准。加州大学伯克利分校认为,Doudna教授与Charpentier教授等人的研究在CRISPR的应用中起到了奠基性的作用,因此Broad研究所获得的专利值得商榷。2016年1月,美国专利及商标局展开了进一步的调查,并于今日做出判决——三名法官认为“nointerferenceinfact”。
业内媒体STAT在一则报道中指出,这短短的四个单词,意味着Broad研究所在2014年获得的关键性CRISPR专利,与加州大学递交的专利申请有足够多的不同。
▲CRISPR相关专利申请一览
“我们递交的专利并非首个与CRISPR应用相关的专利,但它们是首批描述这一发明用于哺乳动物基因组编辑的专利。”Broad研究所在今天发布的一份声明中提到。
值得一提的是,今日的专利判决并不会影响CRISPR-Cas9系统在科学界的应用。作为一家非营利性科研机构,Broad研究所乐于将突破性的发现分享给全球科学界,造福人类健康。因此,Broad研究所也将继续与Addgene(非营利性质粒库)合作,分享这一重要研究工具。自2013年以来,全球59个国家的2000多家研究所已经从Addgene处获得了37000多个与CRISPR-Cas9相关的质粒与试剂。此外,Broad研究所也在今日发表的声明中宣布,将继续为业界的合作伙伴们提供相关研发工具。
张锋博士是麻省理工学院历史上最年轻的华人终身教授。去年,张锋教授作为“下一代领袖”(NextGenerationLeaders)之一,登上了《时代周刊》亚洲版的封面。在报道中,《时代周刊》认为他的工作给CRISPR-Cas9系统带来了巨大变革,让科学家们能够完成先前不敢设想的工作。如今,我们有望能清除每一个受感染细胞中的艾滋病病毒,或是治疗镰刀状红细胞贫血症等经典的遗传疾病。甚至,科学家们已经畅想利用它来攻克癌症的可能。此外,它也能在植物的基因组中得到应用。这能带来全新的生物能源,或带来性状更稳定的作物。
我们期待看到CRISPR-Cas9带来更多有望造福人类的应用!
参考资料:
[1]FORJOURNALISTS:STATEMENTANDBACKGROUNDONTHECRISPRPATENTINTERFERENCEPROCESS
[2]BroadInstituteprevailsinheateddisputeoverCRISPRpatents
[3]张锋教授登上《时代周刊》封面:编辑基因组的下一代领袖
基因敲除是80年代后半期应用DNA同源重组原理发展起来的。80年代初,胚胎干细胞(ES细胞)分离和体外培养的成功奠定了基因敲除的技术基础。1985年,首次证实的哺乳动物细胞中同源重组的存在奠定了基因敲除的理论基础。到1987年,Thompsson首次建立了完整的ES细胞基因敲除的小鼠模型。直到现在,运用基因同源重组进行基因敲除依然是构建基因敲除动物模型中最普遍的使用方法。
2.诱导性基因敲除也是以Cre/loxp系统为基础,但却是利用控制Cre表达的启动子的活性或所表达的Cre酶活性具有可诱导的特点,通过对诱导剂给予时间的控制或利用Cre基因定位表达系统中载体的宿主细胞特异性和将该表达系统转移到动物体内的过程在时间上的可控性,从而在1oxP动物的一定发育阶段和一定组织细胞中实现对特定基因进行遗传修饰之目的的基因敲除技术。人们可以通过对诱导剂给予时间的预先设计的方式来对动物基因突变的时空特异性进行人为控制、以避免出现死胎或动物出生后不久即死亡的现象。常见的几种诱导性类型如下:四环素诱导型;干扰素诱导型;激素诱导型;腺病毒介导型。
科研人员所使用的“基因编辑技术”,顾名思义,能够让人类对目标基因进行“编辑”,实现对特定DNA片段的敲除、加入等。而CRISPR/Cas9技术自问世以来,就有着其它基因编辑技术无可比拟的优势,技术不断改进后,更被认为能够在活细胞中最有效、最便捷地“编辑”任何基因。
一、与诺奖“擦肩而过”的CRISPR/Cas9技术
这不是CRISPR/Cas9这项明星技术第一次得到人们的关注。在此之前,有着“豪华版”诺奖之称的“2015年度生命科学突破奖”颁发给了发现基因组编辑工具“CRISPR/Cas9”的两位美女科学家——珍妮弗·杜德娜和艾曼纽·夏邦杰。二人更是获得了2015年度化学领域的引文桂冠奖——素有诺奖“风向标”之称,曾被认为是今年诺贝尔化学奖的最有力竞争者。
那CRISPR/Cas9到底是一项什么技术,为何能够获得如此这般青睐,又何以在短短两三年时间内,发展成为生物学领域最炙手可热的研究工具之一,并有近700篇相关论文发表?它将来又会如何影响到我们的生活?
CRISPR/Cas9是继“锌指核酸内切酶(ZFN)”、“类转录激活因子效应物核酸酶(TALEN)”之后出现的第三代“基因组定点编辑技术”。与前两代技术相比,其成本低、制作简便、快捷高效的优点,让它迅速风靡于世界各地的实验室,成为科研、医疗等领域的有效工具。
1、过表达目的蛋白,用以检测基因表型;
2、荧光标签标记目的蛋白,用以跟踪其细胞 定位;
3、引入野生型等位基因。
各位同仁大家好,最近在尝试利用CRISPR/cas9技术敲除基因,苦于手里头资源有限,只有lentiCRISPRv2以及相应的包装体系,没有pSpCas9(BB)-2A-Puro(PX459)V2.0,无奈公司货期较久,想立刻开展实验,不知哪位同仁有此质粒并愿意交换,不甚感激。
基因敲除指利用染色体间基因打靶的原理,通过将设计好的打靶载体导入细胞后,同源臂发生同源重组,从而在基因组的某个特定位点引入预定的突变,获得基因型发生了改变的动物。可以分为基因敲入和基因敲出:基因敲入即引入新的基因或突变;基因敲出即使某个特定的基因得到破坏,可以用于研究基因的功能。
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