Problems with co-localization (GFP+lysotracker red) - (May/15/2005 )
Hi,
I am getting very strange results with my co-localization studies in COS cells between a lysosomal protein cloned into GFP (I have only cloned it into C-ter GFP by now), using lysotracker red dye as lysosomal marker. Because the possible lack of especificity, I'm using the recommended concentrations of lysotracker (between 50 and 75 nM with an incubation of 30 min), although I've seen in bibliography one group using lysotracker at 10 mM during 10 min.
The case is that GFP transfection and fixation of cells is working well. Confocal images show diffuse green fluorescence in cytoplasma in negative control GFP transfected cells, as it should be, and localized in numerous and rounded organelles -that seem to be lysosomes- in GFP+wt transfected cells.
Lysotracker red fluorescence is also localized in rounded forms. Lysotracker staining seems to work well.
However, red and green fluorescence don't co-localize at all, moreover, the red "organelles" seem to fill the spaces between the green "organelles". They seem to complementarize but no co-localizate !
I don't know if it is a problem of the GFP or the lysotracker staining. I would like to use antibodies anti LAMP1 or LAMp2 as lysosomal markers but I don't want to spend money in this , if it is possible. I've heard of another group that have had problems with these comercial antibodies and have problems with lysosomal co-localization too.
I will wait ultil I can tranfect with my protein cloned at N-ter of GFP to see if it happens the same.
Do you have any suggestion of what could be happenig, which is the problem or what can I do to?. Has anyone used lysotracker and can give me any advice?
Big thanks!
it may be your filters..are they sub-pixel registered?
I am getting very strange results with my co-localization studies in COS cells between a lysosomal protein cloned into GFP (I have only cloned it into C-ter GFP by now), using lysotracker red dye as lysosomal marker. Because the possible lack of especificity, I'm using the recommended concentrations of lysotracker (between 50 and 75 nM with an incubation of 30 min), although I've seen in bibliography one group using lysotracker at 10 mM during 10 min.
The case is that GFP transfection and fixation of cells is working well. Confocal images show diffuse green fluorescence in cytoplasma in negative control GFP transfected cells, as it should be, and localized in numerous and rounded organelles -that seem to be lysosomes- in GFP+wt transfected cells.
Lysotracker red fluorescence is also localized in rounded forms. Lysotracker staining seems to work well.
However, red and green fluorescence don't co-localize at all, moreover, the red "organelles" seem to fill the spaces between the green "organelles". They seem to complementarize but no co-localizate !
I don't know if it is a problem of the GFP or the lysotracker staining. I would like to use antibodies anti LAMP1 or LAMp2 as lysosomal markers but I don't want to spend money in this , if it is possible. I've heard of another group that have had problems with these comercial antibodies and have problems with lysosomal co-localization too.
I will wait ultil I can tranfect with my protein cloned at N-ter of GFP to see if it happens the same.
Do you have any suggestion of what could be happenig, which is the problem or what can I do to?. Has anyone used lysotracker and can give me any advice?
Big thanks!
Good point on the filters cause GFP excites/emits 3 fold or so less than FITC or something like that. Enhanced GFP is closer but still shy a fold or so and they still photobleach. Anyway, your problem could just be fluorescence intensity. Do you have equal intensities and no colocalization? If you do or don't and want to improve that try a FITC conjugated anti-GFP antibody.
If you do have equal fluorescence then you should look up what lysotracker stains cause it isn't just Lysosomes!
There is also an occasional bleed thru from Lysotracker (LT) to the FITC filter if some condition/filter settings/lumination are incorrect. I read that Zeiss FITC filter has Lysotracker Red listed under it. Strange bec the manufacturer's BS for LT has excitation and emissions that are totally out of the FITC range.
Hope this helps!