Fluctuation assays

Hi Xiaoli,

Thanks for reading our paper and for the questions.

Our choice of drug concentration was based on standard concentrations used in the field for Cryptococcus. The 5-FOA fluctuation assay was previously used (https://doi.org/10.1371/journal.ppat.1002936) in a fluctuation assay for Cryptococcus as cited in the paper. The combination of FK506 and Rapamycin was using the standard concentrations we used in the lab for each individually, but in combination. If you are trying to do a fluctuation assay using a different drug or a different organism, you should determine empirically what concentration is sufficient to inhibit growth while still allowing the possibility of mutations that confer resistance. This should ideally be done in a separate pilot experiment prior to a full-scale fluctuation assay. We did this type of pilot for the combination of FK506/Rapamycin to be sure that we could recover resistant isolates.

You are correct that FALCOR is no longer available online- I was similarly displeased to make that discovery in the past couple years. I unfortunately don't have a back-up to access FALCOR itself, but have successfully used http://shinyflan.its.manchester.ac.uk/ as a replacement since for fluctuation assays.

Thanks,

Blake Billmyre

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