Mitotracker is incorporated into the mitochondria of live cells and retained following formaldehyde fixation.
Although the single mitochondrion in Trypanosoma brucei isn’t as extensive in bloodstream form cells as it is in the insect-stage procyclic form, it can still be easily detected, typically as a single line through the cell body (see image, below).
TbSIR2rp3GFP (green) localises to the mitochondrion (red; stained with Mitotracker) in bloodstream form T. brucei; DAPI staining reveals the nucleus and kinetoplast (large and small blue spots, respectively); scale bar, 5 µm.
- Grow bloodstream form T. brucei to a density of approximately 1×106 cells per ml
- Prepare a 1 mM stock of Mitotracker (Invitrogen/Molecular Probes, #M7512) in DMSO, i.e. 50 μg CMXROS in 94 μl
- Add 1 μl of the stock Mitotracker solution to a 10ml culture, giving a final concentration of 100 nM, and incubate at 37oC for 5 minutes
- Fix cells in 2% PFA in PBS and carry out immunofluorescence, as normal
- Using a higher concentration of Mitotracker or longer incubations results in fragile cells; following immunofluorescence, the mitochondrion, kinetoplast and nucleus can be seen, however the cell body integrity is lost
- The final DMSO concentration in the culture medium should not exceed 0.1% (Eva Gluenz, personal communication); following the above protocol gives a final DMSO concentration of 0.01%
- Mitotracker is provided in 50 μg tubes and should be stored in the dark at -20oC, indefinitely; it is stable in solution for up to 1 month, though we have not tested its effectiveness following solubilisation and storage
- CMXROS is a red dye; hence, any co-staining will require a GFP-tagged protein or FITC-conjugated secondary antibody
- To label the mitochondrion of procyclic form T. brucei, follow the above protocol, but incubate the cells at 27oC for 30 minutes