Cytoprohts .doc

Luminescence HTS
Kit for Cytotoxicity
proliferation
Instructions for use:
CytoPro HTS (6410000)
CytoPro HTS Kit (6410000)
INTENDED USE
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 ASSAY PRINCIPLE
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 KIT CONTENTS
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.
REAGENT RECONSTITUTION
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
HIGHLY RECOMMENDED!
THE OPTIMAL WORKING TEMPERATURE FOR ALL
REAGENTS IS +22°C. ALLOW ALWAYS REAGENTS TO REACH
ROOM TEMPERATURE BEFORE USE.
EQUIPMENT
Instrumentation:
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:
384-well plate
96-well plate
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) PROCEDURAL NOTES
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).
ASSAY PROCEDURE
1. 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.) 2. 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.
3. Cells are treated, according to appropriate protocols, with compounds
inducing cell proliferation or cytotoxicity.
4. Ensure that ATP Monitoring Reagent/ Somalyze solution is stabilized
to room temperature before use, the optimal temperature for reaction 5. 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
Cells/well
Cells/well
HEp2 on 96 well plate
Jurkat on 96 well plate
Cells/well
Cells/well
Figure 1. 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
Figure 2. 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 PRODUCTS
LUMINOSKAN 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 CONSUMABLES
Polystyrene test tubes for tube luminometers, 1000 pc. Bulk LUMINESCENCE KITS
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 LUMINESCENCE REAGENTS
ATP Monitoring Reagent, 250 tests with 96-well microplates or 50 tube luminometric tests Luciferase Substrate, for In vivo eukaryotic luciferase assays, 100 tests MANUFACTURER:
Thermo Labsystems Oy
Fax: +358-9-32910415Internet:http://www.labsystems.fi DATE OF ISSUE:
30.05.2001

Source: http://www.pathtech.com.au/files/03140H7K9N9C3I0B1B8J9G391H27/LuminescenceHTSKit.pdf