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FACS and confocal microscopy issue

FACS confocal microscopy MFI

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#1 pehuen87

pehuen87

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Posted 14 August 2013 - 10:21 AM

Hi all, I would like to ask you about a problem that I´m having.

 

I have two different conditions, let´s say Jurkat #1 and Jurkat #2. My hypothesis is that control cells (Jurkat #1) have normal concentration of PIP2 at plasma membrane, and in treated cells (Jurkat #2) this concentration is diminished.

 

I transduced the cells with a lentivral particle that leads to the expression of the construct PH-GFP (PLC). This protein binds to phosphatidylinositol-4,5-biphosphate (PIP2) and is useful to track this lipid in the cell.

 

When I analysed the median fluorescence intensity of Jurkat #1 and #2 transduced with this lentivirus by FACS, it was exactly the same. I had around 50% of cells transduced and a MFI of approximately 1000. This probably reflects that the expression of the PH-GFP is the same in both cell types.

 

But then, when I saw the cells by confocal microscopy, although I could clearly see the PH-GFP distribution at the plasma membrane of control cells, the visualization of the PH-GFP pattern in the treated cells  (which should be more cytosolic because there´s less PIP2 in PM to be bound by PH-GFP) is almost impossible. The fluorescence of this cells was too low, and unquantifiable (even if the MFI by FACS was the same than control cells!).  These results were obtained using the same laser intensity, same adquisition conditions, etc. Also, the cells for FACS and confocal analysis were the same.

 

My theory is that membrane bound proteins are more concentrated thus being able to be more easily seen. When the protein adquires a cytosolic distribution it gets "diluted" and it becomes harder to see by confocal microscopy.

 

I don´t know... Do you have any ideas? Did somebody have similar problems?

 

Thanks a lot!!

Pehuén.

 







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