| aCella™ - TOX Bioluminescence Non Radioactive Cytotoxicity Assay (GAPDH) |
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| Introduction to aCella-TOX: | ||||||||||||
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Cell Technology introduces aCella-TOX, a new and highly sensitive assay using our patented Coupled Luminescent technology for the detection of cytotoxicity (1). This assay quantitatively measures the release of Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) from Primary Cells, mammalian cell lines, bacterial cells(1,2,3).
Other enzyme release assays(5,6,7) for example the Lactate Dehydrogenase (LDH) release assay (5,6,7), are inconvenient and/or slow and may suffer from low sensitivity as a result of the poor signal and interference by serum or phenol red present in the media. The ATP-release assay (8) is inconvenient and much less sensitive than aCella-TOX, and is unsuitable for use in a cytotoxicity assay because the lytic signal is indirect. aCella-TOX can work in both these media formulations and allows overnight assays while retaining its sensitivity. The sensitivity of aCella-TOX is also greatly enhanced by the coupled luminescent signal-amplification system, which yields a strong luminescent signal from even small amounts of released enzyme. |
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| Assay Principle: | ||||||||||||
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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. |
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The aCella-TOX method has been tested with many modes of cytolysis, including;
The method is highly general, since all known cells express copious amounts of GAPDH, and, unlike other enzymes, GAPDH is very readily released from the cytoplasm upon cell lysis. Using specially adapted formulations, the sensitivity of the method can be driven below 1 eukaryotic cell (2), which is impossible with any other reported liquid-phase method. Please consult with us if you have an application requiring specialized techniques.
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| Use of aCella-TOX for Measurement of Cell-Mediated (T Cells, ADCC, NK) or Complement-Mediated Cytolysis | ||||||||||||
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Unlike virtually all standard assays, including 51Cr release and the Eu3+ assays, aCella-TOX does not require labeling of the target cells. No separations are needed. After completion of the lytic process under study, the aCella-TOX reagent is formulated and added to the wells, and luminance is read after 3-5 minutes. Due to the extreme sensitivity of aCella-TOX, especially if serum-free or heat-killed media are used, it is frequently possible to shorten the incubation time for the lytic process. It is usually possible and desirable to use smaller numbers of T cells than are needed for the 51Cr-release assay, due to the high sensitivity of aCella-TOX and the fact that excessive numbers of effector cells can increase the background signal.
Citations
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
Ogbomo H, et.al - Histone deacetylase inhibitors supress natural killer cell cytolytic activity - FEBS Lett (2007). Click here to read the publication
Identification of polymerase and processivity inhibitors of vaccinia DNA synthesis using a stepwise screening approach - Janice Elaine Y. Silverman, Mihai Ciustea, Abigail M. Druck Shudofsky, Florent Bender, Robert H. Shoemaker and Robert P. Ricciardi - Antiviral Research, In Press - June 20 2008
Resistance to Cytarabine induces the Up-regulation of NKG2D Ligands and enhances Natural Killer (NK) cell Lysis of Leukemic Cells - H Ogbomo, Martin Michaelis, Denise Klassert, Hans Wilhelm Doerr and Jindrich Cinatl Jr. - Neoplasia, Vol 10, No 12, pp 1402-1410, Dec 2008 Inhibition of human mesenchymal stem cell-derived adipogenesis by the environmental contaminant benzo(a)pyrene - Normand Podechard, Olivier Fardel, Michel Corolleur, Marc Bernard, and Valérie Lecureur - Toxicology In Vitro - Vol 23, Issue 5, Aug 2009 - In Press 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 |
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aCella™ - TOX Bioluminescence Non Radioactive Cytotoxicity Assay (GAPDH)/生物发光非放射性细胞毒性检测（磷酸甘油醛脱氢酶）
主要优点：
安全--- 非放射性酶释放检测
多功能---可以用来检测T细胞，原代细胞，NK的活性，和其他的细胞溶解酶试剂。可以用添加血清的培养基进行检测
一步，免洗。可以用样品板检测
快速--- 3-5分钟可以完成。铬51或者铕的释放用来检测非常耗时。荧光素酶检测的内在灵敏性会随着扩增效率而提高，从而产生大量高能量的分子。
高灵敏性--- 可以检测少于500个细胞/孔（血清存在的情况下）或者10个细胞/孔（无血清培养基或者热灭活培养基）
GAPDH: GAPDH是细胞中的天然成分，所以不需要以任何形式将其引入细胞中。与其他检测方法相区别，该检测方法需要预先标记的细胞，转染，或者用其他方法将蛋白加入到靶细胞中，使其产生信号用于下一步实验。
细胞介导的或补充介导细胞溶解检测的优势 – 可以使用更少数量的T 细胞，比铬51的量要少 - 释放检测，因为过多的效应细胞可以增加背景信号。可能是由于aCella - TOX高灵敏度的原因。
ADCC / CMC检测 - 非放射性检测，用来替代铬51。
HTS – 适用于高通量模式
非破坏性检测，需要其他参数检测。
aCella-TOX的介绍:
Cell Technology 介绍了aCella - TOX，这是一种新的高灵敏度的用于细胞毒性检测的技术，该技术已经获得了专利（1）。用于原代细胞，哺乳动物细胞系，细菌细胞的甘油醛-3-磷酸脱氢酶（GAPDH）的定量检测（1,2,3）。
其他酶的释放试验（5,6,7），例如乳酸脱氢酶（LDH）释放检测法（5,6,7），该方法不方便或比较慢，并由于低灵敏度，使结果产生弱信号，使结果不可信。培养基中的血清或酚红会给实验带来干扰。而ATP释放检测法（8）不方便，灵敏性远低于aCella - TOX，并且因为裂解信号是间接，不适用于检测细胞毒性。
aCella - TOX可以过夜检测，同时保留其灵敏度。当偶联了发光信号—扩增系统，aCella - TOX灵敏度可大大增强，从而产生出强烈的荧光信号（即使在少量的酶的释放的情况下）。
检测原理:
GAPDH是糖酵解和糖异生途径的重要的酶。这种同源四聚体酶在辅助因子和无机磷存在下，催化D -甘油醛-3 -磷酸氧化磷酸化成1,3 - 二磷酸甘油酸。
在aCella – TOX反应体系中， GAPDH的释放与3 - 磷酸甘油磷酸激酶（PGK）相联系，并产生ATP。 用荧光素酶检测ATP---荧光素生物发光。此外，aCella – TOX可以在同一块板上检测细胞毒性和细胞活力。培养的上清液可以从原来的板子上去掉，并在不同的板子上检测。该检测方法没有破坏性，可以检测更多的参数，如基因表达。
