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CFSE staining of mycobacteria - (Sep/30/2009 )

Hi all!

I'd like to track mycobacteria in the mouse for at least 2 weeks and therefore would like to stain them with CFSE. Does anyone have experience with that? I would like to know the CFSE concentration and how long to stain them.
We have a staining protocol for DCs that uses 1uM CFSE for 10min RT and then 2 washes. But I guess I would need to stain longer/ use a higher concentration for the mycobacteria and the long time frame? I don't expect them to divide a lot but I don't know how fast the CFSE will fade anyway.
Also the mycobacteria have all this thick and sticky cell wall and I'm not sure how well the CFSE would come through. And I need to fix the cells with formalin (or ethanol or PFA) before facsing for security reasons. Does that affect CFSE fluorescence?

I could do some concentrations and times and FACS the cells to assess staining but to be sure they're still alive, CFU would take >3weeks and I'm actually quite eager to get the real experiment started.

So I would appreciate any help with that!



try this for staing:
after centrifugtion>resuspend your cells in 2x PBS>incubate 20 l from the sample in ur staining (1g/l> for 15 min at RT> WASH 2times with 2x PBS
Good luck