FAQs

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Frequently asked questions will be available soon for our Hyphen BioMed products. Currently we have FAQ's available for the Xenometrix product line.

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AMES MUTAGENICITY ASSAYS FAQ

Why should I perform the Ames II / Ames MPF Mutagenicity Assays rather than the standard plate incorporation test?

With the ready-to-use reagents and media, media preparation, autoclaving and sterility testing is no longer necessary. The Ames II / Ames MPF Mutagenicity Assays are liquid microplate modifications of the standard Ames test which offer a higher speed format, easier handling and the possibility of automated plating and plate reading. The assays are fast and efficient, show good correlation with the standard Ames test and consume less test compound and consumables than the standard Ames test.

Do you offer presentations of your tests in our facilities?

We are happy to discuss your requirements and make suitable arrangements for you. Please contact us directly to discuss this.

How long does it take to perform the Ames II / Ames MPF Mutagenicity Assays?

The assays last two and a half days, however with much less hands-on-time as compared with the standard Ames test.

How much test compound is needed?

With a top dose of 5 mg/ml, 6 concentrations, triplicate, a ½ log dilution range, 2 strains +/- S9, the lowest amount of test compound is 26.5 mg. With a top dose of 1 mg/ml, 6 concentrations, triplicate, a ½ log dilution range, 2 strains+/- S9, the lowest amount of test compound is 5.3 mg.

Which bacterial strains are available for the Ames II / Ames MPF Mutagenicity Assays?

In the Ames II Salmonella typhimurium TA98 is used for the detection of frameshift mutations and TAMix for the detection of base pair mutations. TAMix is a mixture of the strains TA7001, TA7002, TA7003, TA7004, TA7005, TA7006 in equal proportions. Individually these strains are designed to revert by only one specific base-pair substitution. With this mixture, all the possible base pair substitutions can be represented in one culture. In the Ames MPF series of mutagenicity tests the following S. typhimurium strains recommended by the OECD guideline 471 are available: TA98, TA100, TA1535, and TA1537. Two E.coli strains also mentioned in the guideline 471 are also available in the Ames MPF format: E. coli wp2 uvrA and E. coli wp2 [pKM101].

Are there publications with the Ames II / Ames MPF assays?

Please refer to our "Literature" section. (Link)

Can we send you compounds for testing?

Yes, we do test compounds for customers with any of our strains or their combination. A Secrecy Agreement may be signed for commercially sensitive information and materials.

Are the strains, reagents and media available separately?

Yes, all the necessary components for performing the Ames II /Ames MPF Mutagenicity Assay are also available separately.

How many compounds can I test with your kits?

The Ames II / Ames MPF Mutagenicity Assays are available in 2 sizes with enough bacteria and media for either 1 or 10 samples. All kits allow to test the indicated number of samples in triplicate at 6 concentrations, with negative and positive controls.

Can you provide S9 / positive control chemicals / plastic ware?

We also provide S9 fraction and positive controls, and if needed the necessary plastic ware for the 1-sample kits. (For reasons of cost effectiveness we do not sell larger amounts of plastic ware.)

Sometimes when I am scoring my 384-well plates, there are wells that look like they are starting to turn from purple to yellow, but are not completely yellow. Should these be scored as positive or negative?

Any color change away from the purple of negative wells should be considered positive. Often (but not always) a small colony of bacteria is visible in positive wells. We recommend to use a light box in order to better see the changes in color and the colonies.

 

CYTOTOXICITY ASSAYS FAQ

I have problems with bubbles in the wells. They give me unreliable OD readings.

It is essential to remove all bubbles before reading the OD of the wells. This can be achieved by passing a gas flame over the wells. You can use an inverted Bunsen burner or a mini torch. Be careful not to overheat the wells or to burn the plastic. Be careful for the presence of flammable materials or liquids next to your workspace. You could also use a hair dryer for this purpose.

I observe strong variations of OD readings between replicate wells.

Possible sources of variation are: cell clumps resulting in uneven cell numbers in the wells; partial loss of cells during washing steps; uneven pipetting of reagents (loose tips). Be particularly careful for the presence of air bubbles in your wells. Remove them with a gas flame (Caution!) or a hair dryer.

What solvents up to what concentration can I use in the tests?

This depends also on the cell line and the length of incubation. If your substance is not soluble in water we recommend to try DMSO. It is safe to use in most systems up to 2%. If higher concentraionts need to be used you should first test the solvent effect compared to a blank. Ethanol or methanol can be used as a solvent, tool. Again, 2% should be fine.

Glucose test:

Do I have to use such strong acids like 12N H2SO4 or 37% HCI at the end of the Glucose test?

Yes, an optimal color development requires the use of strong acids.

In the Glucose test I see very little difference between the medium control and the positive control with cells. What can I do to increase this difference?

First, make sure you dilute your samples when using media with high glucose content. The assay can measure glucose concentrations in the range of 1 - 200 μg/ml.

You can increase the lenght of incubation of the cells and/or the number of cells per well. It can take several days until cells have consumed 50% of the available glucose. As a rough indication you can observe the color of the medium: when it turns yellow the cells have most likely also consumed a significant part of the total glucose.

The reconstituted glucose working solution has turned slightly pink after storage at 4°C for several weeks. Can I still use it?

Yes, as long as no precipitate has formed you can still use the solution. The absolute OD readings will be slightly higher, but the OD differences should be about the same.

LDH test:

Can I perform the kinetic OD reading also at RT?

Yes, the kinetic reading can also be done at RT. You should extend the reading to 1 hr.

My plate reader does not allow to read at 340 nm. Can I use an alternative wavelength setting?

No, you need a plate reader capable of reading plates at 340 nm.

Why is there a slow decrease of OD340 WIth time even in the medium control?

Fetal Calf Serum used in the medium is a (usually small) source of LDH activity. If this activity is too high, you may try to reduce the amount of FCS in your medium or switch to another FCS source.

Can I store the supernatant and run the assay at a Later time?

Yes, you can store the plate at 4°C for a few hours, provided it is sterile and you have a protein source in your medium (FCS, BSA). We do not recommend freezing of the supernatant. Be sure the supernatant and the assay plate have room temperature before performing the assay.

What kind of cytotoxic substances can be measured by this test?

The test is particularly suitable for substances affecting the intergrity of the cell membrane.

Can I directly compare the results (units per ml) obtained with results obtained with a different LDH assay?

No, enzymatic activities measured as units per volume depend on experimental conditions (e.g. buffer composition, temperature, substrate concentrations). If you want to compare results with an other test you could run the same substance in both tests and see how the results correlate.

Pyruvate is often present in culture media such as Dulbecco's, Iscove's or Ham -F12. In the test kits of competitors it can interfere with the assay - does it also interfere in the LDHe assay by Xenometrix?

In the Xenomtrix version of the assay the conversion of pyruvate to lactate by LDH is measured. A defined amount of pyruvate is added to the reaction mixture which is about 50x higher than the contribution by the medium. Therefore, pyruvate present in culture media does not significantly contribute to the reaction equilibrium or reaction rate.

PAC test:

PAC IV Stop Solution has developed a dark precipitate. Can I still use it?

You can remove the precipitate by centrifugation at 3000 rpm for 5 minutes and sill use PAC IV.

XTT / MTT tests:

XTT and MTT both detect mitochondrial metabolism. Which test should I choose and why?

XTT has two main advantages over MTT: It is more sensitive, and the reaction results in a soluble formazan dye. This eliminates a final solubilization step which means less manipulation and consequently a reduced risk of error.

MTT has the advantage of being less expensive.

Another consideration may be the filter settings available with your plate reader: XTT requires 480 or 450 nm plus a reference filter at 690 nm; MTT requires 540 nm plus a reference filter at 690 nm.

If available filters are no constraint in your lab we recommend to use the XTT test.