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GFP expression - microscopy, spectrofluorimetry (Sep/01/2006 )

Hi,

I have transfected my cell line with a GFP encoding vector. Now I can observe fluorescence in the cells by fl.microscopy by exxcitation at 488nm. The fluorescence is diffuse (not very clear) cos my cells are very very small and appear as colonies; so its rather colony fluorescence than individual cells. When I do a spectrofluorimetry analysis the difference between the values of transfected and non-transfected controls is not very high. Now why am I getting this??? Thanks in advance

-smartsunny-

QUOTE (smartsunny @ Sep 1 2006, 10:52 AM)
Hi,

I have transfected my cell line with a GFP encoding vector. Now I can observe fluorescence in the cells by fl.microscopy by exxcitation at 488nm. The fluorescence is diffuse (not very clear) cos my cells are very very small and appear as colonies; so its rather colony fluorescence than individual cells. When I do a spectrofluorimetry analysis the difference between the values of transfected and non-transfected controls is not very high. Now why am I getting this??? Thanks in advance

Can you post a picture to help us to get a sense of what you are talking about?
spectrofluorimetry analysis is not particularly sensitive for EGFP. I know people soemtime have to pool whole dish together to get a reading. FACS is more sensitive and can be quantivative as well, but may cost you money to run in some institutes.

-genehunter-1-

QUOTE (genehunter-1 @ Sep 1 2006, 11:34 AM)
QUOTE (smartsunny @ Sep 1 2006, 10:52 AM)

Hi,

I have transfected my cell line with a GFP encoding vector. Now I can observe fluorescence in the cells by fl.microscopy by exxcitation at 488nm. The fluorescence is diffuse (not very clear) cos my cells are very very small and appear as colonies; so its rather colony fluorescence than individual cells. When I do a spectrofluorimetry analysis the difference between the values of transfected and non-transfected controls is not very high. Now why am I getting this??? Thanks in advance

Can you post a picture to help us to get a sense of what you are talking about?
spectrofluorimetry analysis is not particularly sensitive for EGFP. I know people soemtime have to pool whole dish together to get a reading. FACS is more sensitive and can be quantivative as well, but may cost you money to run in some institutes.


Genehunter, these cells hav not been reported (published report) as yet.. we spoke abt it in the immunology forum too abt surface antigens. Thats why i haven't attached a picture. The size is around 400 - 600 nm diameter. I didn't nkow that we may need to pool a whole dish!!! I used just one well from a 6-well plate. Maybe thats why... I'll try to attach a picture. Hey tnx for ur patience..

-smartsunny-

Hi there,

colleagues of mine have performed titration curves with EGFP-tagged virusses and compared FACS with fluorometry and the signal in the most positive well (meaning about 100% infected cells, suspension cells they were, so there were quite a lot in the wells from a 96-well plate) was indeed not so much bigger than in negative control. Don't remember the exact numbers (because it was felt that this way was useless for our purposes and never repeated), but indeed I would pool a large number of cells and maybe you can get results, but FACS is way more sensitive.

-vairus-