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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 51 Cr – 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 Cr 51 assays. Sample Protocol published.
  • 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(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.

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.
 
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
 
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
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 Content:
  1. Component 1: 4x Enzyme Assay Reagent.............................Part# 6001
  2. Component 2: 1x Enzyme Assay Diluent ..............................Part# 3008
  3. Component 3: Glyeraldehyde 3-Phosphate (G3P)................Part# 6003
  4. Component 4: 50x  Detection Reagent..................................Part# 6002
  5. Component 5: 5.5x Detection Assay Diluent........................Part# 3009
  6. Component 6: Lytic Agent....................................................Part# 3035
The following kits are available:
Catalog #
Size* Price
CLATOX 100-3
500 $375
CLATOX 100-4 1000 $745
* Please call 888 7 ASSAYS (888-727-7297) or email info@celltechnology.com for volume pricing
 
 Product Data Sheet

 Protocol (ADCC / CMC Assay protocol included)

 MSDS