Do I need a licence for using GreenScreen HC or BlueScreen HC?
No. Gentronix has negotiated licences with Amersham Biosciences (now GE Healthcare) and Prolume Inc. to use the GFP and Gaussia Luciferase genes respectively in our assays. These allow us to distribute our products worldwide. Individual end users do not need to obtain a separate licence to use GreenScreen HC or BlueScreen HC products within their own laboratories, and need only sign a straightforward end user label licence before using supplied kit materials.
Contract Research Organisations wishing to offer the GreenScreen HC assay as a service to customers should contact Gentronix for more details since certain licence conditions exist for use of GreenScreen HC that CROs need to be aware of.
There are no restrictions on the use of BlueScreen HC for CROs wishing to provide this assay as a service to customers
Can I have a demonstration of the assay in my laboratory?
When kits are purchased by a user for the first time, Gentronix will usually supply our scientists to run training and installation of the assay in their laboratory. This includes hands on demonstration of the assay, optimization of laboratory equipment especially microplate readers and flow cytometers, and providing training in data processing and interpretation. If you are simply interested in seeing the assay run in situ we also welcome visitors to our laboratory in Manchester, UK. Please contact us to discuss your requirements.
Why do you recommend that I need a site visit for installation and training in your assays?
We want your first experience of GreenScreen HC or BlueScreen HC to be a good one and to make it work first time in your hands; hence we prefer to visit you at your laboratories to guide you through the first set of assays. We appreciate that you employ very competent scientists, but we have learned during our international assessments of transferability and years of experience, that like any new assay, a period of familiarisation is needed, and this is speeded by having an expert on hand. During the visit we would explain and demonstrate the assay protocol, run a series of standard compounds and collect and interpret the data. Importantly we would also set up and optimise protocols for your microplate reader and flow cytometer if appropriate, advise on the automation of the assay if liquid handling robotics are to be used, address any compound specific issues and, if requested, give a presentation about our assay technology to interested members of your staff.
All the companies we have visited have found this approach to be valuable in minimising both their time and effort required to get started with GreenScreen HC or BlueScreen HC and the amount of ongoing technical support subsequently required.
Can I have a free sample?
We do not provide reagents free of charge, however we do adopt a flexible and cost effective approach to enable customers to access our genotoxicity assays via reagent kits or contract service. Please contact us to discuss your specific requirements.
Can we send some compounds to Gentronix for testing?
Yes, we can test compounds for customers in our laboratories at Gentronix as a contracted service. Both catalogue and proprietary compounds can be tested, using ‘blind’ studies if necessary. We receive compounds and can test them in any combination of the assays we provide in accordance with our standard protocols. For GreenScreen HC or BlueScreen HC we usually request just a 10 mg pre-weighed sample of each compound, from which we will test the compound over 9 serial dilutions (8 serial dilutions for BlueScreen) from a maximum concentration of typically 1 mg/ml, unless limited by solubility. We will also run a repeat test if the concentration range initially chosen shows excessive cytotoxicity, compound precipitation or autofluorescence, in order to obtain an unambiguous result. Upon completion of the testing we will provide a report that will give you a summary of the assay features, details of the protocol performed and discussion of the results. We aim to complete the testing within 2 weeks from receipt of compound.
For other Assays the required amount of material may be different to GreenScreen HC or BlueScreen HC. Please contact us to discuss your assay requirements and compound availability.
Importantly, we can deal with both small and large numbers of compounds, again please contact us to discuss your needs.
How many compounds can I run with a reagent kit?
GreenScreen HC and BlueScreen HC come in standard 20 compound kits. The throughput is principally determined by the liquid-handling used for preparation, i.e. manual pipettes or robotic. The minimum run size is 4 compounds (i.e. one microplate) yet up to 40 compounds can be tested by manual pipette in one day, using one microplate reader.
Customers who require large numbers of kits may like to consider technology access as an alternative to kit purchase or larger kit sizes may be considered. Please contact us to discuss your requirements.
How long does it take to do the assay?
It depends on the liquid-handling method used. A multi-probe robotic platform may take 5 – 10 minutes to prepare an assay plate for GreenScreen HC, i.e. serially dilute test compounds, add standard compounds and then add the two cell strains. Whilst by hand using programmable multi-channel pipettes it takes around 20 minutes per plate. The GreenScreen HC assay requires two reading time points at 24 and 48 hours.
How much compound does it use?
This depends on the maximum concentration you wish to test. Usually this is either a fixed amount, or the limit of cytotoxicity or solubility. The assay requires only approx. 0.5 ml of test solution per test. To test from a concentration of 1 mg/ml, if the compound is soluble in the aqueous diluent (2% v/v DMSO) then 1 mg or less of test sample is sufficient. If the compound is to be pre-solubilised in DMSO then typically 10 mg of sample is required.
What compound dilution series is used?
The assay has been optimised to run 4 compounds per microplate, each over 9 concentrations (GreenScreen) or 8 concentrations (BlueScreen), usually using serial 50/50 dilutions. The assay software recognises and interprets data from the defined microplate layout. This wide dilution series also allows the user to capture the toxicologically relevant concentration range in a single run, without the need for a range finding experiment. The magnitude of dilution across the microplate can be altered, however the number of dilutions can not.
What solvents can I use?
The usual solvent is aqueous DMSO (2% v/v for GreenScreen HC), however, others such as pure water or ethanol (4% v/v) can be used. Thus the assay is suited to test compounds that are stored or pre-solubilised in DMSO.
What about compounds that require metabolic activation?
Can I use S9? As eukaryotic cells, the TK6 cells (GreenScreen HC) are equipped with some components of human metabolism such that some pro-mutagenic compounds known to require metabolic activation can be detected using GreenScreen assays. However, like the other in vitro genotoxicity test systems, these cell lines lack the inherent metabolic competency of the liver. GreenScreen HC can be used with S9 using a flow cytometer to collect the data and protocols are available. BlueScreen HC is also compatible with Metabolic activation using S9 fraction.
In addition Gentronix also offers access to Ames and MNT testing which can both be performed with Metabolic activation.
Can I automate the GreenScreen assay on my liquid handling robot?
Yes, GreenScreen HC is designed for use with both handheld multi-channel pipettes and liquid handling robots. If a moderately high throughput is required, i.e. greater than 20 compounds / day, the use of robotic liquid handling to set up the assay microplates may be a more convenient option. We have used a variety of commercially available single and multi-probe liquid handling robots to produce assay plates, including those supplied by BioTek, Hamilton and Tecan.
How long can I passage the TK6 cell lines for GreenScreen HC and BlueScreen HC?
The cell lines are fully validated and guaranteed for up to 3 months of continuous culture after which fresh reagents can be obtained from Gentronix. A second set of aliquots of frozen cell cultures are supplied with each kit in case a replacement is required due to a loss of cell lines during the three month period, due for example to mishandling or breakdown of incubation equipment. Each GreenScreen kit comes with Gentronix’s Quality Control assurance specific to the batch of cell culture and media supplied.
What is the performance with autofluorescent compounds?
By making use of a second, non-fluorescent strain the GreenScreen HC assay protocol is able to accurately correct for the autofluorescence properties of the majority of test compounds. For highly autofluorescent compounds a patented protocol using fluorescence polarisation can also be used to discriminate between the GFP-derived assay signal and compound autofluorescence, exploiting the high fluorescence anisotropy of GFP.
Alternatively, because BlueScreen HC uses luminescence as the light detection mode, problems with autofluorescence are rare.
What is the performance with coloured compounds?
The GreenScreen software alerts the user if the compound is significantly absorbing at the wavelengths used. For most coloured compounds tested to date, the concentration at which the toxic range of the compound is detected by the assay is sufficiently dilute as to present few problems. For highly coloured compounds, such as chemical dyes, a separate experiment diluting the dye in the absence of cells is able to correct up to an optical density of ~5.0 OD units.
Again, with BlueScreen HC we have shown that the confounding effect of coloured compounds is much reduced.
Can I test my compounds with low aqueous solubility?
To aid the analysis of compounds with low aqueous solubility, the assay can be run with a final concentration of DMSO of 1% v/v in GreenScreen HC and 1% v/v in BlueScreen HC, allowing the testing of samples stored or pre-solubilised in DMSO.
Is the automatic data analysis software included in the kit, and what does it cost?
Yes, the data analysis software which produces a detailed report on the genotoxicity and cytotoxicity of each set of 4 compounds per microplate is supplied with the assay kit at no extra cost. As periodic software updates become available these will be sent to all current GreenScreen and BlueScreen users.
What is the comparison of the GreenScreen assay with other tests?
Results from the published validation studies show that in comparison with other test data GreenScreen demonstrates high specificity in the detection of genotoxic carcinogens without compromising sensitivity. Hence, a positive result in GreenScreen is highly predictive of a positive result in the regulatory test battery. Results are highly reproducible using a standardised, validated protocol. GreenScreen detects genotoxic chemicals with a wide variety of mechanisms of action, including those with specific eukaryotic targets, and a different but overlapping spectrum of compounds to bacterial assays.
GreenScreen HC, being a human cell-based assay detects a wide spectrum of compounds. Specifically: Aneugens, since these don’t damage DNA, but mis-segregate whole chromosomes.
Can you show me some typical results?
Example reports are available, please contact us for more information.
Can you give me examples of the classes of genotoxic compounds GreenScreen detects?
The following are examples of genotoxic compounds detected with GreenScreen HC.
- Direct acting genotoxins: Busulfan, Cisplatin, Ethyl nitrosourea, MNNG, 4-NQO, ICR-191
- Topoisomerase inhibitors: Camptothecin, Doxorubicin, Etoposide.
- Aneugens: Benomyl, Colchicine, Griseofulvin, Paclitxel, Vincristine sulphate.
- Nucleotide analogues: 5-Azacytidine, 5-Fluorouracil, Azidothymidine (AZT).
- DNA synthesis inhibitors: Aphidicolin, Hydroxyl urea, Pyrimethamine.
- Reactive oxygen species generators: Bleomycin sulphate, Hydrogen peroxide.
Besides GreenScreen HC and BlueScreen HC, what other assays does Gentronix provide?
Ames MPF, Comet, MicroFlow MNT, Flow Cytometry