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aCella™ - TOX
Bioluminescence Non
Radioactive Cytotoxicity Assay (GAPDH)
Promo! - SAVE upto $50 in November 09 - Scroll below
for details
|
| 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
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
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: |
- 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
|
| Kit Content: |
- Component 1: 4x Enzyme Assay
Reagent.............................Part# 6001
- Component 2: 1x Enzyme Assay Diluent
..............................Part# 3008
- Component 3: Glyeraldehyde 3-Phosphate
(G3P)................Part# 6003
- Component 4: 50x Detection
Reagent..................................Part# 6002
- Component 5: 5.5x Detection Assay
Diluent........................Part# 3009
- Component 6: Lytic
Agent....................................................Part# 3035
|
| The
following kits are available: |
Catalog #
|
Size* |
Promo Price |
CLATOX 100-3
|
500 |
$455 |
| CLATOX 100-4 |
1000 |
$895 |
|
| * Please call 888 7 ASSAYS
(888-727-7297) or email info@celltechnology.com
for volume pricing |
|
|
