aCella™ - TOX
Bioluminescence Non Radioactive Cytotoxicity Assay (GAPDH)
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.
Introduction to aCella-TOX:

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 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.

Assay Principle:

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.

Applications:
The aCella-TOX method has been tested with many modes of cytolysis, including;
  • cellular cytotoxicity (T cells)
  • complement (2,3), pore-forming agents,
  • antibiotic-mediated lysis of bacteria, and
  • detergent mediated and mechanical lysis
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.
Use of aCella-TOX for Measurement of Cell-Mediated (T Cells, ADCC, NK) or Complement-Mediated Cytolysis

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.

aCella™-TOX


Figure 1: 5000 Ramos cells/well were incubated with serially diluted Rituxan antibody for 15 minutes prior to the addition of purified NK cells stimulated overnight with IL-2. The ADCC reaction was further incubated for 2 hours at the specified E:T ratios. % Cytotoxicity was measured using the aCellaTOX assay.

aCella™-TOX


Figure 2: 5000 Ramos cells/well were incubated with serially diluted Rituxan antibody for 15 minutes prior to the addition of purified NK cells stimulated overnight with IL-2 . The ADCC reaction was further incubated for 3.5 hours at the specified E:T ratios. % Cytotoxicity was measured using the aCellaTOX assay. 3.5hour timepoint has been optimized with the 10:1 E:T ratio

aCella™-TOX



Figure 3: Figure: The above graph shows an antibody titration of Rituximab from0.000977 to 1ug/ml incubated with 5% baby rabbit complement and 5000 Daudi target cells for 30 minutes at 37oC

aCella™-TOX


Figure 4: 5000 Daudi cells/well were incubated with serially diluted Rituxan antibody for 15 minutes prior to the addition of purified NK cells stimulated overnight with IL-2. The ADCC reaction was further incubated for 2 hours at the specified E:T ratios. % ADCC was measured using the aCella-TOX assay.Log (EC50) value for E:T 25:1 was -2.344 and for E:T 10:1 was -2.213

aCella™-TOX


Figure 5: A Direct comparison of 51Cr and aCella-TOX was carried out with the same donors with Daudi Cells. Log (EC50) value for aCella-TOX was -2.23, and that for 51Cr was -2.087 for Donor A.

aCella™-TOX


Figure 6: A Direct comparison of 51Cr and aCella-TOX was carried out with the same donors with Daudi Cells. Log(EC50) value for aCella-TOX was -2.612, and that for 51Cr was -2.77 for Donor B.


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Product Data Sheet
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Protocol (ADCC / CMC Assay protocol included)
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MSDS
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ADCC Assay 51Cr V/S aCella-Tox Comparison
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A Novel Bioluminescent Assay to Measure ADCC
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

Tumor cells infected with oncolytic influenza A virus prime natural killer cells for lysis of resistant tumor cells - Henry Ogbomo, Martin Michaelis, Janina Geiler, Marijke van Rikxoort, Thomas Muster, Andrej Egorov, Hans Wilhelm Doerr and Jindrich Cinatl Jr - Medical Microbiology and Immunology - 15 December 2009

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 - Pravin T.P. Kaumaya, Kevin Chu Foy, Joan Garrett, Sharad V. Rawale, Daniele Vicari, Jennifer M. Thurmond, Tammy Lamb, Aruna Mani, Yahaira Kane, Catherine R. Balint, Donald Chalupa, Gregory A. Otterson, Charles L. Shapiro, Jeffrey M. Fowler, Michael R. Grever, Tanios S. Bekaii-Saab, William E. Carson, III - - Journal of Clinical Oncology, Vol 27, No 31 (November 1), 2009: pp. 5270-5277

A novel immunomodulatory mechanism of ribavirin in suppressing natural killer cell function - Henry Ogbomo, Martin Michaelis, Behric Altenbrandt, Hans Wilhelm Doerr, and Jindrich Cinatl Jr - Biochemical Pharmacology, Vol 79, issue 2, Jan 2010, pp 188-197

A Distinct Subset of Proinflammatory Neutrophils Isolated from Patients with Systemic Lupus Erythematosus Induces Vascular Damage and Synthesizes Type I IFNs - Michael F. Denny, Srilakshmi Yalavarthi, Wenpu Zhao, Seth G. Thacker, Marc Anderson, Ashley R. Sandy, W. Joseph McCune, and Mariana J. Kaplan - The Journal of Immunology, 2010, 184, 3284 -3297

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

Role of the Innate Immune Response and Tumor Immunity Associated with Simian Virus 40 Large Tumor Antigen - Devin B. Lowe, Michael H. Shearer, Joel F. Aldrich, Richard E. Winn, Cynthia A. Jumper and Ronald C. Kennedy - Journal of Virology, October 2010, pp 10121-10130, Vol. 84, No. 19

The Development of Live-Attenuated Vaccines for Pandemic Influenza - Catherine J. Luke and Kanta Subbarao – National Institute of Allergy and Infectious Diseases, NIH – Infectious Disease, 2010 , Vol 3, Part 12, 423-430, doi: 10.1007/978-1-60761-512-5_42

The anti-tumoral drug enzastaurin inhibits natural killer cell cytotoxicity via activation of glycogen synthase kinase-3β - Henry Ogbomo , Tsigereda Biru , Martin Michaelis , Nadine Loeschmann , Hans Wilhelm Doerr, and Jindrich Cinatl Jr. – Biochemical Pharmacology, Vol 81, Issue 2, 15 Jan 2011, pp 251-258. doi:10.1016/j.bcp.2010.09.026

A variant human IgG1-Fc mediates improved ADCC - Ross Stewart, George Thom1,Michaela Levens, Gülin Güler-Gane, Robert Holgate, Pauline M. Rudd, Carl Webster, Lutz Jermutus and John Lund - Protein Engineering, Design and Selection (2011) doi: 10.1093/protein/gzr015 First published online: May 18, 2011. Published by Oxford University Press

Bi-specific Aptamers Mediating Tumor Cell Lysis - Achim Boltz, Birgit Piater, Lars Toleikis, Ralf Guenther, Harald Kolmar and Bjoern Hock - The Journal of Biological Chemistry, 286, 21896-21905. doi: 10.1074/jbc.M111.238261 First Published on April 29, 2011. Published by The American Society for Biochemistry and Molecular Biology, Inc.

Autologous CTL response against cancer stem-like cells/cancer-initiating cells of bone malignant fibrous histiocytoma - Masanobu Kano, Tomohide Tsukahara, Makoto Emori, Masaki Murase, Toshihiko Torigoe, Satoshi Kawaguchi, Takuro Wada, Toshihiko Yamashita, Noriyuki Sato – Cancer Science, Vol 102, Issue 8, pp 1443-1447, August 2011.

Spontaneous antibody, and CD4 and CD8 T-cell responses against XAGE-1b (GAGED2a) in non-small cell lung cancer patients - Yoshihiro Ohue, Shingo Eikawa, Nami Okazaki, Yu Mizote, Midori Isobe, Akiko Uenaka, Minoru Fukuda, Lloyd J. Old, Mikio Oka, Eiichi Nakayama – International Journal of Cancer – Jan 2012.

References:
  1. Methods and compositions for coupled luminescent assays. United States Patent 6,811,990 Corey and Kinders, issued November 2, 2004.
  2. 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
  3. 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.
  4. 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.
  5. 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.
  6. 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
  7. 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.
  8. Korzeniewski, C. and Callewaert, D.M. (1983) An enzyme-release assay for natural cytotoxicity. J. Immunol. Meth.64, 313-20.
  9. 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.
  10. 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
Kit contents:
1. Component 1: 4x Enzyme Assay Reagent.............................Part No. 6001
2. Component 2: 1x Enzyme Assay Diluent ..............................Part No. 3008
3. Component 3: Glyeraldehyde 3-Phosphate (G3P)................Part No.6003
4. Component 4: 50x Detection Reagent..................................Part No. 6002
5. Component 5: 5.5x Detection Assay Diluent........................Part No. 3009
6. Component 6: Lytic Agent....................................................Part No. 3035
6. Component 7: 5 Lumi Plates (Catalog# CLATOX100-3L)
6. Component 8: 5 Lumi Plates + 5 Tissu Culture Plates (Catalog# CLATOX100-3P)

 ProductCatalog #Size*Price
Non Radioactive Cytotoxicity Assay- aCella-TOXCLATOX 100-3500 $745.00
Non Radioactive Cytotoxicity Assay- aCella-TOX (with 5 Lumi Plates)CLATOX 100-3L500$795.00
Non Radioactive Cytotoxicity Assay- aCella-TOX (with 5 Lumi Plates + 5 Tissue Culture Plates)CLATOX 100-3P500 $845.00
Non Radioactive Cytotoxicity Assay- aCella-TOXCLATOX 100-41000$1,445.00

* Please call 888 7 ASSAYS (888-727-7297) or email info@celltechnology.com for volume pricing