I am transfecting my zebrafish ES cell line with a GFP tag plasmid. Besides counting GFP cell by using hemocytometer, is there any way to calculate the transfection efficiency?
We use to check the efficiency of cell transfection with GreenFP or RedFP plasmids by FACS analysis. The software reports the % of fluorescent cells/total cells. It is very simple... having the instrument
microscopic observation will give you an idea about the efficiency for GFP tagged plasmid ,and also you can do FACS analysis for quantitative measure .
Hi
My suggestion depends on the fate of your cells (to be lysed or not). The efficiency will on its part will depend on the size of your GFP tag plasmid and much more.
Encode with a reporter gene like luciferase and run a luciferase assay for luciferase activity after your transfection - but there is often cell lysis in this method. :( hope it helps.
Gilbert
Dear guys, thanks for the suggestion!
Shakuntala Mahilkar: If counting the GFP cells through microscope, if there any easier way to count, because counting thousands of cells is very tiring. Thank you!
We use to check the efficiency of cell transfection with GreenFP or RedFP plasmids by FACS analysis. The software reports the % of fluorescent cells/total cells. It is very simple... having the instrument
@ Gen Jen Yang ....hey u get it wrong my point was ...you can just have an idea about the the transfection efficiency by observing it under microscope you can say that aprox 50% or 80% of your cells get transfected.just by observation no need to count the cells...yes for exact counting i suggested the FACS ...you can ask Paola Di Bonito for its detail.
I don't have FACS here, so I think the only easy way is to count cells by using hemocytometer. Thanks for the advises and ideas!
you should compared and count cells observed in phase contrast with those fluorescent observed in the same photo and counted under uv light . You can sample several fields of your plate, correcting the value for the surface of the plate, you should have a good approximation
% transfection efficiency = (number of cells stained with fluorescent positive control dye/total number of cells per field) X 100
It is recommended use multiple fields per well and do at least 3 different wells at the same condition to get statistically significant efficiencies.
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