Luminescent ATP Detection Assay Kit - ab113849 - Instructions for Use For the rapid and quantitative detection of ATP This product is for research ...
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ab113849 – Luminescent ATP Detection Assay Kit Instructions for Use For the rapid and quantitative detection of ATP This product is for research use only and is not intended for diagnostic use.
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Table of Contents
1. Introduction 3
2. Assay Summary 5
3. Kit Contents 6
4. Storage and Handling 6
5. Additional Materials Required 7
6. Assay Procedure 8
7. Data Analysis and Sample Data 12
21. Introduction
Total levels of cellular ATP can be used to assess cell viability, cell
proliferation and cytotoxicity of a wide range of compounds and
biological response modifiers. This kit irreversibly inactivates ATP
degrading enzymes (ATPases) during the lysis step, ensuring that
the luminescent signal obtained truly corresponds to the endogenous
levels of ATP. The major advantages to this luminescence assay
are:
1. Long luminescence signal: The half life of the luminescent
signal is greater than 5 hours and therefore a special
luminometer with injectors is not required.
2. Fast: results are obtained in less than 30 minutes.
3. Simple: there are no separation steps and there are only 2
reagent additions.
4. Homogenous: No cell harvesting or centrifugation required.
5. Sensitive: the limit of detection is 5 cells in 100µL.
6. Wide linear dynamic range: approximately from 1pM to
1µM of ATP (this will vary depending on instrument
sensitivity).
3While total levels of cellular ATP are a hallmark of cell viability,
proliferation, cytotoxicity and cell death under standard culture
conditions they are not representative of the mitochondrial
bioenergetic state of the cell. Cultured cells can survive in the
absence of an active electron transport chain by supplying all their
ATP demands through glycolysis. However, when cells are cultured
with non-fermentable carbon substrates such as galactose, they
require an active electron transport chain (ETC) to supply all their
ATP demand. Under these conditions, damage to the mitochondrial
bioenergetic state of the cell will lead to depletion of cellular ATP,
which can be easily measured by luminescence as a decrease in
total levels of ATP measured by kit ab113849.
Principle: The ATP assay kit is an adenosine 5’-triphosphate (ATP)
monitoring system based on firefly (Photinus pyralis) luciferase. This
assay is used to determine the total levels of cellular ATP in culture.
The ATP assay is based on the production of light caused by the
reaction of ATP with added luciferase and D-luciferin. The emitted
light is proportional to the ATP concentration inside the cell. The
reaction can be summarized as follows:
ATP + D-Luciferin + O2 Oxyluciferin + AMP + PPi + CO2 + Light
Mg2+
LUCIFERASE
4Limitations:
• FOR RESEARCH US ONLY. NOT FOR DIAGNOSTIC
PROCEDURES.
• Use this kit before expiration date.
• Do not mix or substitute reagents from other lots or sources.
• Any variation in operator, pipetting technique, washing
technique, incubation time or temperature, and kit age can
cause variation in binding.
2. Assay Summary
Prepare a 96 well plate with cells and biological modifiers in a
total volume of 100µL
Add 50µL of detergent, Incubate 5 minutes
Add 50µL of substrate solution, Incubate 5 minutes
Read
5For the provided ATP standard (optional) - Reconstitute the ATP
standard and prepare an ATP dilution series. Add 10µL of each ATP
dilution to wells containing 100µL of media + 50µL of detergent,
incubate, add 50µL of substrate solution, incubate 5 minutes, read.
3. Kit Contents
• Detergent : 20 mL
• Substrate Buffer: 20 mL
• Lyophilized substrate: 3 vials
• Lyophilized ATP standard: 1 vial
4. Storage and Handling
Store all components at 4°C in the dark. Once the lyophilized
substrate is reconstituted as a substrate solution, its activity will
decline to 30% of original levels after 1 week of storage at 4°C.
Reconstituted substrate solution may be stored at -20°C for longer
periods of time. After thawing, crystals may appear in the buffer.
These crystals can be dissolved by warming the vial at room
temperature and mixing the contents. Reconstituted ATP standard is
stable for weeks when stored at -20°C. Diluted ATP solutions are
stable for eight hours when stored on ice.
65. Additional Materials Required
• Luminometer.
• General tissue culture supplies
• Sterile, tissue culture treated, white or black 96-well
microplates.
• 50 – 300μL Multichannel pipettor
• ATP-free dispensing materials
76. Assay Procedure
ATP ASSAY PROTOCOL
A. Grow cells and seed on a 96-well plate
1. Prepare a 96-well plate with cells that have been treated
with growth factors, cytotoxic agents or other type of
biological modifiers. Do not exceed 100µL per well (see
Table 1 for suggested cell line specific seeding numbers). If
working with different cell lines from those mentioned in
Table 1, determine in advance the optimal seeding by cell
titration.
Table 1: Cell line specific seeding number:
Cell Line Seeding/well
HELA 12,000 cells/well
HepG2 25,000 cells/well
HdFn 12,000 cells/well
SH-SY5Y 50,000 cells/well
2. Include blank wells (without cells) to determine the
background luminescence in your system and positive
control wells to determine normal signal expected (e.g.
8untreated or vehicle treated culture). If a standard curve is
necessary for interpretation of results, ensure that you leave
enough empty wells on the plate to add the ATP standard as
shown in section B.
B. ATP standard
In cases where it is necessary to quantify the amount of
ATP present in the sample, perform the following steps in a
96-well plate in parallel to section C.
1. Reconstitute the vial of lyophilized ATP standard solution
with deionized water to make a 10mM stock solution (i.e. if
the amount printed on the label is 12µmol add 1200µL of
water). Vortex the vial for one minute until fully dissolved.
2. Prepare an ATP standard dilution series with deionized
water in microfuge tubes ranging from approximately 10µM
to 100pM. Keep diluted standards on ice. These dilutions
are stable for 8 hours on ice.
3. Pipette 100µL of complete culture medium into the empty
wells of the plate allocated for the standard in section A.
4. Add to each well 50µL of the detergent and shake the plate
for five minutes in an orbital shaker at 700rpm (in parallel to
step 2 and 3 from section C).
95. Add 10µL of the ATP dilution series to the wells and shake
the plate for five minutes in an orbital shaker at 700rpm
(before step 5 from section C).
6. Add to each well 50µL of the reconstituted substrate solution
and shake the microplate for five minutes in an orbital shaker
at 700rpm (in parallel to step 5 from section C).
7. Dark adapt the plate for ten minutes (in parallel to step 6
from section C).
8. Measure luminescence.
10C. ATP procedure
Work with subdued lighting, out of direct sunlight or direct
bright fluorescent lighting. Bright light may cause plate
phosphorescence resulting in higher background levels.
Phosphorescence has a half-life of several minutes.
1. Take all the components from the kit and allow them to
equilibrate to room temperature.
2. Add 50μL of Detergent to the 100μL of media in each well.
3. Shake the plate for five minutes in an orbital shaker at 700
rpm. This lyses the cells and stabilizes the ATP.
4. While plate is shaking, reconstitute each vial of lyophilized
substrate with 5mL of substrate buffer.
5. Add 50μL of the reconstituted substrate solution to the wells
and shake the microplate for five minutes in an orbital
shaker at 700rpm.
6. Dark adapt the plate for ten minutes.
7. Measure luminescence.
117. Data Analysis and Sample Data
Subtract background (empty wells) from all test measurements and
either: (1) determine ATP levels as percentage from positive control
or (2) interpolate test measurements from the ATP standard curve.
Data in Figure 1 shows the luminescent counts obtained in HepG2
cells after 4 hours of treatment with rotenone and vehicle control.
20,000
Luminescent counts
15,000
10,000
5,000
0
ne
l
ro
no
nt
co
e
ot
e
R
cl
µM
hi
Ve
25
4
Figure 1. ATP detection kit cytotoxicity assay result. 2.5x10 HepG2
cells were seeded into each well, allowed to adhere and treat for 4 hours
with 25µM rotenone and vehicle control (DMSO) in glucose based complete
media. After treatment, cells were lysed, exposed to the ATP substrate
solution and signal was measured on a luminescent counter. Mean and
standard deviation is plotted for 3 replicates from each condition. Rotenone
induces cytotoxicity in HepG2 cells.
12This assay may be used for cytotoxicity screening of developmental
compounds. A suggested microplate template is depicted below (fig.
2) to screen 3 compounds in triplicate dose response per plate.
Figure 2: Suggested assay template. In this assay example, rows A and
H as well as columns 1 and 12 are not seeded with cells and are left with
media only. Rows A and H will help determine the background
luminescence. Columns 1 and 12 will be used to set up the ATP standard in
a 1:5 dilution series from 1µM to 12pM final concentration. Wells in column
c 2
are used as the diluent control wells to determine the maximal expected
expe
signal in the absence of compound.
1314
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