I have tried to stain Leishmania mitochondria with a mitotracker but I am not getting clean staining for mitochondria.
I have uses the following protocol for staining:
1) 1 micro liter of 100 micromolar mitotracker stain was added to 999 micro liter culture of leishmania.
2) Incubation at 37 degree for 15 minutes.
3) Wash the cell with serum free media.
4) incubation at 37 degree for 15 minutes.
5) Wash with 1X PBS.
6) Fix the cell with 2% PFA for 10 minutes.
7) Wash the cells with 1X PBS (2 times).
I have even checked cells before fixation but the staining was not specific to mitochondria. I am getting background staining as well.
Hi Durgesh.
I've not used mitotracker in Leishmania, but I have stained bloodstream form Trypanosoma brucei mitochondria using the protocol described at the link below. I've found that the quality of staining is very sensitive to the amount of mitotracker used and the duration of the incubation - you may need to optimise both these parameters; these may also be influenced by the particular life cycle stage that you're working with. Hope this helps.
http://blogs.lshtm.ac.uk/alsfordlab/protocols/mitotracker-staining/
Thank you very much Sam! I think I am using high concentration of mitotraker. I will try this protocol with lesser concentration of mitotraker. I appreciate your generosity of sharing this information with me, once again thank you very much.
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