I want to do a bioassay with yeast cells and S9-mix for metabolic activation to determine the toxicity of pro-mutagens. Are there any other protocols than the Ames-test for the use of S9-mix, because I want to test it in liquid medium for 24 h?
The Ames ll test is done in liquid medium and can be combined with S9 activation. You can find more information at Xenometrix (http://www.xenometrix.ch) where you also can purchase a complet kit.
I've used the S9 mix as Xenometrix (the'original one' in Boulder, US) on a number of their assays and it works reasonable in the way they perform it. Be aware however that S9 mix come with a heavy load of protein and color formation in your assay. One immediate side effect is color in colorimetric assays, you do get a significant increase in background. A second problem is that you don't know what all the protein is doing in terms of bioavailability and cell interferences. In many instances we've seen growth arrest in E. coli. At the moment we are working on a protocol to get rid of both issues by doing the S9 activation without cells, followed by gel filtration. After that we dose the cells. Still waiting for results however.
Good points Freddy and interesting new developments. I can confirm that test substance binding and bioavailability are important aspects in this assay, at least for hydrophobic organic chemicals. We have thus recently combined this assay with passive dosing, and this study showed that the dosing really can make a difference in this assay:
https://www.researchgate.net/publication/230878862_The_dosing_determines_mutagenicity_of_hydrophobic_compounds_in_the_Ames_II_assay_with_metabolic_transformation_Passive_dosing_versus_solvent_spiking?ev=prf_pub
Article The dosing determines mutagenicity of hydrophobic compounds ...
Dear Stephanie,
My lab developed S9 protocols for use with a mammalian cell cell, TK6. S9 is quite toxic, so we only include the S9 plus cofactors, for 3 hours exposure. It is then washed away using centrifugation, and the cells allowed to grow in fresh medium for another 45 hours. The wash is required because S9 is quite toxic, and as Freddy mentioned, S9 is colouored so careful washing remves this interferences if you have a colorimetric endpoint. During the recovery time they may either correctly repair any DNA damage, or fix a mutation. Yeasts generally divide quicker than mammalian cells, so you could probably use a shorter recovery time - do a few time courses to identify best exposure/recovey times. I have attached our paper, describing the human cell S9 assay. I hope this helps a little!.
Article A pre-validation transferability study of the GreenScreen HC...
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