flourescence in non-transfected cells! - (Jun/18/2008 )
I am using Sf9 cells and have transfected them with GFP plasmid. We don''t have an inverted flourescence microscope in my lab. So to observe the cells, I resuspended them in PBS and placed them on a slide and observed them under a normal phase contrast microscope in Uv mode. I was able to observe the flou. The problem is I could detect the flourescence even in the control (cells that were not transfected) cells. Why am I seeing this??? Is it normal - do these cells have some auto-flourescent properties or is this common with all cell types???
Thanks in advance for all your suggestions and explanations....
Some cells do. I know primary hepatocytes have high autofluorescence. Many dead cells also have this false positive signal. The level is usually weaker thna EGFP. If you fix these cells, it becomes worse.
Thanks Genehunter... Any suggestions on how to differentiate between cells that are expressing fluorescence due to GFP and those due to autofluorescence in the transfected group?
You can tell the difference by the intensity between transfected vs control group. If your scope has the adjustment for exposure time, use it . Otherwise, you have to use immunostain against EGFP to be sure.
Thank you. Will explore that.... still not very sure whether the filter has a broad window.