I recently tested the components of the "Yeast vacuole staining kit" from Life technologies for staining vacuoles in Penicillium chrysogenum. Carboxy-DCFDA and MDY-64 (both use the GFP filter) gave acceptable results. However, none of the CMAC-based dyes (CellTracker Blue CMAC, CMAC-Arg, CMAC-Ala-Pro) worked... These would be especially useful to me as I would prefer to use the DAPI filter installed in my microscope.
Does anybody have some experience for using these vacuole markers in filamentous fungi? Help would be greatly appreciated!
CMAC works very well in yeast at a final concentration of 2µg ml-1...not sure about the rest!
Dear Christian,
I will also test same kit for Hansenula polymorpha cells. I want to know how can I adjust cell number based on OD levels (i.e. how much cells should be collected)?
Thanks in advance,
Arman
Hello Arman!
There is a nice overview on OD 660 nm values and cell numbers here:
http://www.pangloss.com/seidel/Protocols/ODvsCells.html
Please keep in mind that this table shows data for S. cerevisiae. But I think it should serve well as a template for H. polymorpha cells.
In the attachment you will find the manual for this kit. You will need 10^6 cells/mL so a 1/10 dilution of cells at OD 0.680 (corresponding to 1*10^7 cells/mL) should work fine. Hint: use 50 mM sodium citrate buffer, pH 5, for the dilution.
Success with your experiments!
Best regards, Christian
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