high basal levels of fluorescence in flow cytometry - (Jul/07/2014 )

I need help with a problem with my cells sh-sy5y

I have a stable transfected cell line (they were seleccionated with G418, and they are keeping with G418 all time). When I measure fluoresence for Bgtx-FITC in flow cytometry, I have higher levels of fluorescence in my control cells (non- transfected cells without Bgtx) than my transfected cells (of course, without Bgtx).

Can G418 affect to basal fluorescence? Can g418 remove the autofluorescence?

How is possible  they are different basal fluorescence if they are the same cells?

Thanks

martin-sanchez on Mon Jul 7 16:27:08 2014 said:

I need help with a problem with my cells sh-sy5y

I have a stable transfected cell line (they were seleccionated with G418, and they are keeping with G418 all time). When I measure fluoresence for Bgtx-FITC in flow cytometry, I have higher levels of fluorescence in my control cells (non- transfected cells without Bgtx) than my transfected cells (of course, without Bgtx).

 

Can G418 affect to basal fluorescence? Can g418 remove the autofluorescence?

 

How is possible  they are different basal fluorescence if they are the same cells?

 

Thanks

 

Absolutely. Aminoglycosides inhibit peptide synthesis. However, it probably wouldn't block it to the extent that your flow data suggests.

More likely it is a technical issue.

 - It is still odd that you get a FITC+ signal in your control that is stronger than your transfected cells. Does your unstained control have the same FITC levels as your stained control?

 - I'm assuming your washed after staining, and you gated out doublets and other noise during analysis.

 - In that case, there may be some non-specific binding going on, perhaps by some scavenging Fc receptors.

 - Maybe you could post your FCS files for scrutiny.

If you're sure that your FC was done correctly, then I would answer your question by running a Western to see if your protein is still there.

Thanks for your answer.

My stained cells of control (non -transfected) have higher fluorescence than my unstained control cells, so that, I have a good fluorescence signal. The same happens with my transfected cells, but with intensity very different.

If G418 affects to basal fluorescence, do you recommend me that I remove G418 before I do the experiment?  how many time?   four days before?

I use FCS during the incubation with Bgtx-FITC to avoid non -specific binding. After I wash cells.