I want to stain a microorganism with a sterol specific fluorescent-dye for FACS and reculture high sterol producers after sorting.
I know the fluorescent probe Filipin is available for staining sterols. But will my cells remain alive after sorting?
If probably not, would there be another probe available, more suited for the job?
Hi,
Filipin is a commonly used fluorescent probe for staining sterols, and it can be used to identify cells or microorganisms that have high levels of sterols. However, one concern with using this probe is that it can be toxic to cells and may affect their survival after staining and sorting.
There are some other fluorescent probes that have been used to identify sterols in living cells, such as filipin III and ergosterol analogs. These probes are less toxic than filipin and may allow the cells to survive sorting.
However, it's important to keep in mind that any staining procedure of live cells can have a negative effect on their survival and subsequent growth, so it is always recommended to test the effects of staining and sorting on cell survival and growth before starting any experiments.
Additionally, if your goal is to only reculture high sterol producer cells, you could consider sorting based on an indirect method. For example, you could use a reporter system where a gene that is known to be upregulated in cells with high levels of sterols is linked to a fluorescent protein, allowing you to sort on this reporter rather than on the sterol levels directly.
It's also important to note that depending on the specific microorganism you are working with, it's not always possible to find a fluorescent probe that is completely non-toxic and can preserve the cell viability. In those cases, you may have to settle for a trade-off where you'll be able to sort based on the sterol content but the cell viability will be reduced.
In any case, it is always best to consult the literature or experts in your field to see what methods have been used successfully for your specific microorganism before starting your experiment.
Regards.
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