Dear All,
I have a couple of GFP tagged proteins that I want to study for localization in Mycobacteria. Which stains should be utilized for this purpose (if the cell wall to be stained). Can anyone please provide the detailed protocol?
Thanks in Advance
Have a good day!!
Regards
Renu Bisht
Hi Renu,
I am not aware of membrane properties of mycobacteria, but as I work with plants, and some microbes, I have experienced several issues with sold-as "membrane permeant" dyes.
Those I tested successfully either on microbes and plants, and that are suitable with GFP detection :
DNA stains : Syto Orange 81, 83, 84 ; DRAQ5 (highly toxic and expensive).
Respiratory chain stain : CTC (I don't know wether mycobacteria have respiratory chains)
Transmembrane potential accumulation : Mitotracker Orange or Red, DASPMI, and other mitochondiral probes work well on Rhizobia. I suppose it is the same for your mycobacteria. There are some non-fluorescent reduced forms of Mitotrackers that accumulate in mitochondrai/bacteria, and become fluorescent when oxidized by respiratory chain.
I used Mitotrackers at concentrations ranging from 5 to 50 nM, Syto Orange dyes at 5 µM. Maybe you should make tests to be sure you use the right amount of dye.
For the protocol, the simpler, the better : just add the dye in the suspension at fixed OD, let the dye stain for 10 min and read with the flow cytometer.
Hi Olivier,
Thanks for your detailed reply. Mycobacterial membrane is lipid rich so I was thinking of staining those cell wall lipids with auramine and rhodamine dyes. I am not sure whether these stains can interfere with the GFP signal on visualization under confocal microscope. I am not planning any FCM for this purpose as of now.
I can surely read about mitotrackers if they can be utilized for this work.
Thanks and Regards
Renu Bisht
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