Kit for Cytotoxicity
Instructions for use:
CytoPro HTS Kit (6410000)
The CytoPro HTS kit is intended for rapid assay of cytotoxicity and cell
proliferation using bioluminescent ATP technology. The kit is formulated
to work for high throughput screening in 96 and 384 well microplates
and it offers a one step homogenous assay suitable for mammalian cell
lines in culture. The CytoPro HTS Kit can be used for the direct
assessment of cell numbers, substitute for tritiated thymidine uptake or
MTT assays as a measure of cell proliferation, and also to assay for
cytotoxic effects on cultured cells. It may therefore be used to replace
conventional endpoint measurements in the determination of either
agonist or antagonist activity on specific cell lines, e.g. in cytokine
The kit is based on the bioluminescent measurement of ATP which is
present in all metabolically active cells. The bioluminescent method
utilises an enzyme, luciferase, which catalyses the formation of light
from ATP and luciferin according to the following reaction:
ATP + D-Luciferin + O > Oxyluciferin + AMP + PP
The emitted light intensity is linearly dependent on the ATP
concentration and is measured using a luminometer. Bioluminescence is
now the most widely used method for the assay of ATP due to its very
high sensitivity, wide dynamic range, and ease of use.
The CytoPro HTS Kit offers many advantages over conventional methods
by avoiding the use of radioisotopes, giving greater reproducibility and
by being very rapid (the results are available within few minutes). The kit
has been formulated to be used with Thermo Labsystems Luminoskan
1. ATP Monitoring Reagent (Yellow cap), lyophilized, 2 vials
2. Somalyze (Red cap), 2 x 50 ml
The ATP Monitoring Reagent should be stored at -18°C. Somalyze may
be stored at +4°C or lower. See kit carton for expiry date of the whole kit.
See bottle labels for expiry date of individual components.
ATP Monitoring Reagent/Somalyze solution:
Reconstitute the lyophilized ATP Monitoring Reagent with Somalyze.
Add 10 ml of Somalyze into the glass vial (yellow cap), replace the
rubber stopper and mix gently. When the lyophilized ATP Monitoring
Reagent is completely dissolved, transfer the solution back to the
Somalyze bottle. Rewash the glass bottle once again with 10 ml
combined reagent and return the liquid back to the plastic bottle (red
cap). This results in 50 ml of combined reagent that is sufficient for the
assay of five complete 96 or 384 well microplates. Allow the reagent to
equilibrate for 15 minutes at room temperature.
Reconstituted reagent stability: +25°C: 8 hours, +4°C: 5 days
TAKE GREAT CARE WHEN HANDLING ANY OF THE
REAGENTS. SKIN HAS HIGH LEVEL OF ATP ON ITS SURFACE
WHICH CAN CONTAMINATE THE REAGENTS LEADING TO
FAULTY HIGH READINGS. WEARING LATEX GLOVES IS
THE OPTIMAL WORKING TEMPERATURE FOR ALL
REAGENTS IS +22°C. ALLOW ALWAYS REAGENTS TO REACH
ROOM TEMPERATURE BEFORE USE.
The kit has been formulated for Thermo Labsystems Luminoskan Ascent
Microplate Luminometer with one automatic dispenser but can also be
used with Thermo Labsystems Fluoroskan FL. Dispenser is primed with
combined ATP Monitoring Reagent/Somalyze solution and set to
dispense 25 µl or 100 µl for 384 or 96 well plates, respectively. The
instrument is programmed to dispense combined reagent into each well,
and after 5 minutes ATP releasing period to take an 280 ms or 1000 ms
integrated reading for 384 or 96 well plates, respectively. The complete
assay can be automatized with Ascent software provided with
Luminoskan Ascent and Fluoroskan FL.
The following protocol allows for 5 minutes ATP releasing period when
all the wells of 384 or 96 well plate is used.
Protocol for the instrument:
NOTE: KEEP ALWAYS THE LUMINOMETER DISPENSING LINES CLEAN.
Additional Equipment and Consumables:
a) White (or white clear bottom) tissue culture treated 96 or 384 well
microplates suitable for luminescence measurements.
b) Thermo Labsystems ATP Standard, Product number 6415200.
c) Thermo Labsystems Multichannel micropipettes (8-Channel Cat no.
4510 030 and 16-Channel Cat no. 4510 070) and sterile disposable
tips for transferring the cell solutions (8-Channel Cat no. 9400 263)
1. The kit contains all the required reagents to assay 10 complete
microplates. The recommended culture volume per well is 25 µl in
384 well plates and 100 µl in 96 well plates. Modifications to the
protocol may be necessary if larger culture volumes are used.
2. Before cellular ATP can be measured by bioluminescence it must first
be extracted from the cells. An extraction reagent, Somalyze is
supplied with the kit. To increase the throughput of this assay, the ATP
Monitoring reagent is added together with Somalyze, allowing a one-
3. Extraction of ATP is allowed to occur for 5 minutes and the amount of
ATP of the extracts is then measured automatically. Extraction time
can be optimized for each cell type, but as a general rule 5 to 10
minutes is optimal for adherent cells while shorter times can be used
for suspension cells (for example 2.5 minutes).
The eukaryotic cells are grown in appropriate media and are divided in
optimal cell population to a white (or white clear bottom) tissue
The assay is suitable for both adherent and suspension cell
The optimal cell population can be defined for a particular cell
line. In general, cell densities from 50 to 25 000 or 200 - 50 000
can be used on 384 or 96 well plates, respectively. (Fig. 1.)
It is recommended that the total cell sample volume in the wells is 25
µl for 384 well plates and 100 µl for 96 well plates. Modifications to
the protocol may be necessary if larger culture volumes are used.
Cells are treated, according to appropriate protocols, with compounds
inducing cell proliferation or cytotoxicity.
Ensure that ATP Monitoring Reagent/ Somalyze solution is stabilized
to room temperature before use, the optimal temperature for reaction
After required incubation time load the microplate into the
luminometer and initiate the protocol as described in the equipment
section. The luminometer will automatically add 25 µl or 100 µl of the
ATP Monitoring Reagent/Somalyze solution to each well of 384 or 96
well plate, respectively, and measure the light emission after 5
HEp2 on 384 well plate
Jurkat on 384 well plate
HEp2 on 96 well plate
Jurkat on 96 well plate
Varying amounts of cells were plated on 384 (A and B) or 96 (C and D) well
plates. Cells were allowed to recover for 24 hours before measuring with Thermo
Labsystems Luminoskan Ascent luminometer. Values represent average of 16 (A and B) or
INTERPRETATION OF RESULTS
In most assays, utilizing cell proliferation or cytotoxicity to determine the
activity of growth factors such as cytokines, the direct luminometer
output in Relative Light Units (RLUs) are used to calculate the desired
activity (directly analogous to using cpm in radioisotope based assays).
For high throughput screening of compounds inducing cytotoxicity or
cell proliferation, untreated cells can be used as a control. Accordingly,
the output in RLUs of untreated cells (RLU
effect of cytotoxic compounds is calculated by comparing to this value:
Figure 2 shows a sample of cytotoxic effect of etoposide on K-562 and
Different culture media may quench the light output from the
bioluminescent reaction to differing degrees. For example phenol red at
5 mg/l concentration, which corresponds to the amount in RPMI culture
medium, decreases RLU to approximately half form the values without
phenol red. There may also be differencies in the efficiency of Somalyze
to release ATP from different cell types.
If preferred, results can be expressed in terms of cellular ATP
concentration. The ATP Standard can be used to generate a standard
curve to which all samples can then be compared. Due to the wide
dynamic range of the ATP assay, the standard curve may cover the range
from 10-9 M to 10-6 M for 384 well plates and from 10-10 M to 10-6 M for
96 well plates in the reaction mixture. The reaction mixture used for
standard curves should consist ATP dilution in the appropriate fresh
complete culture medium and the assay should be performed
correspondingly to actual cell samples.
Effect of etoposide on
HEp2 and K-562 cells
. 1000 cells/well were plated on 384 well plates and allowed to attach overnight.
Cells where then treated with 100 µM etoposide or DMSO (control) for 48 hours before
measuring with Thermo Labsystems Luminoskan Ascent luminometer. Results represent
LUMINESCENCE PRODUCTSLUMINOSKAN ASCENT (FOR MICROPLATES)
Luminoskan Ascent 100-240V with one dispenser
LUMINOSKAN TUBE LUMINOMETERS
Luminoskan TL Plus, Generation II, 100 - 240 V
Luminoskan TL Plus Portable Monitoring Unit*) Availability will be informed later
Polystyrene test tubes for tube luminometers, 1000 pc. Bulk
Quantitative ATP Monitoring Kit, for free ATP, 1250 tests
CytoPro Kit, for cytotoxicity and cell proliferation assays, 500 tests
GenGlow 100 Kit, for eukaryotic luciferase expression assays, 100 tests
GenGlow 1000 Kit, for eukaryotic luciferase expression assays, 1000 tests
ATP Lysis and Assay Kit, for tissue samples, 50 tests
ATP Biomass Kit, for microbial ATP detection from aqueous samples, 200 tests
Greenlight Kit, simplicity for hygiene monitoring, 20 tests
ATP Monitoring Reagent, 250 tests with 96-well microplates or 50 tube luminometric tests
Luciferase Substrate, for In vivo eukaryotic luciferase assays, 100 tests
Thermo Labsystems Oy
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