Hello everybody,
I want to analyze yeast cells in flow cytometry (excitation 488 nm) by use of a fluorescence indicator (GFP). I've got the problem, that autofluorescence of yeast cells is higher than the single of my fluorescence dye. So I thought about, how to minimize the autofluorescence of yeasts. Or would a plasmolysis of the cells (removal of intracellular parts) would be a solution for such problems.
Could you tell me your opinion about this topic?
Thanks in advance.
1. avoid using YPD media and switch to SDC.
2. if you are working with a ade- strain, you will have an autofluoresence issue du to accumulation of phosphoribosylaminoimidazole. So, you can add extra adenine to your media to avoid the accumulation of that fluorescent intermediate.
Check your construction, are you sure the GFP is expressed? You can verify with a microscope if you have fluorescence.
I agree with Miguel, don't use YPD media.
You also can wash your cells with PBS before your FACS experiment.
Thanks for your reply-.
My construct is correct and GFP is expressed. I verified by fluorescence spectroscopy. Tomorrow I will check by fluorescence microscope how it is visible.
I wash my cells with PBS five times without sucess for flow cytometry measurements. Instead I get more fragments with each additional washing step (more events at low FSC signal)
What composition of SDC can you recommend??
Marco. You can use this protocol for synthetic media preparation
https://aem.asm.org/content/81/22/7813
There I also wanted to avoid auto fluorescence in yeast for microfluidics analysis
You can consider changing the GFP to a bright red dye like mScarlet. You normally have less problems with autofluorsecence in this channel
I made some fluorescence microscopic measurements and saw a very less signal of GFP. But in flow cytometry measurements a still have big signals of yeast autofluorescence. I also avoided YPD and tested the SDC medium but signals were as big as before, so I have a overlap of GFP and autofluorescence signals. Is there maybe a possibility to enhance the GFP signals in solution?
The primary issue is that you chose (or had to use) GFP as a reporter in a high autofluorescence cell type. GFP is neither that bright, or expressed (typically) well enough post-transfection (as you saw in microscopy) to differentiate from autofluorescence.
I would highly recommend, if possible, to switch to a reporter than can be excited by a red laser, and that is as bright as possible out of all red excitable dyes. Autofluorescent molecules have lowest emission in red spectrum.
As long as your autofluorescence signal is greater than your GFP, any quenching reagents will be useless, as they would equally quench GFP as well as autofluorescent molecules. Plasmolysis of the cell, as far as I understand, would remove liquids, rpimarly water, from within the cell. Autofluorescence is primarily from proteins and membranes, thus plasmolysis would not be effective.
Honestly, i would really go back to transfecting with a different reporter, high autofluorescence and low GFP signal is a classic issue with no real solution but to select a very bright reporter for the red laser.
which promoter are u using? i'm sick of making gfp fusion proteins and rarely i had such problems. did u check by western blot that u indeed are expressing gfp? because maybe u just have a wrong clone...
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