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| Caspase
/ Apoptosis Detection |
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High
Throughput Screening
- Apo3HTS |
|
Caspase
Poly,1,2,3,7,8,9,10 Detection - FAM-FMK |
|
Caspase
Poly, 1,2,3,8,9,10, Detection
- (SR-FMK) |
|
Antibody
Specific Caspase 3 Detection
- Apo Active 3 (FITC) |
|
Antibody
Specific Caspase 3 Detection
- Apo Active 3 (PE) |
|
| NOS/ROS
Detection Kits |
|
|
| Fluorescent
Enzymatic Assays |
|
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| Mitochondria
Membrane Potential detection |
|
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| aCella
- Bioluminescence Assays Kits |
|
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| Cytotoxicity |
|
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| ELISA
Products |
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| Cathepsin
B,K,L Detection Kits |
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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 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* |
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 |
|
|
