{"id":809,"date":"2013-09-11T16:48:38","date_gmt":"2013-09-11T15:48:38","guid":{"rendered":"http:\/\/blogs.lshtm.ac.uk\/alsfordlab\/?page_id=809"},"modified":"2014-11-26T11:30:03","modified_gmt":"2014-11-26T10:30:03","slug":"mitotracker-staining","status":"publish","type":"page","link":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/mitotracker-staining\/","title":{"rendered":"Mitotracker staining"},"content":{"rendered":"

Mitotracker is incorporated into the mitochondria of live cells and retained following formaldehyde fixation.<\/p>\n

Although the single mitochondrion in Trypanosoma brucei<\/em> isn\u2019t 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).<\/p>\n

\"\"<\/a>
\nTbSIR2rp3GFP<\/sup> (green) localises to the mitochondrion (red; stained with Mitotracker) in bloodstream form T. brucei<\/em>; DAPI staining reveals the nucleus and kinetoplast (large and small blue spots, respectively); scale bar, 5 \u00b5m.<\/p>\n

Alsford et al<\/em> (2007) Mol Microbiol<\/em> 63<\/strong>:724-36<\/a>.<\/p>\n

 <\/p>\n

 <\/p>\n

Protocol<\/strong><\/em><\/p>\n

    \n
  1. Grow bloodstream form T. brucei<\/em> to a density of approximately 1×106<\/sup>\u00a0 cells per ml<\/li>\n
  2. Prepare a 1 mM stock of Mitotracker (Invitrogen\/Molecular Probes, #M7512<\/a>) in DMSO, i.e. 50 \u03bcg CMXROS in 94 \u03bcl<\/li>\n
  3. Add 1 \u03bcl of the stock Mitotracker solution to a 10ml culture, giving a final concentration of 100 nM, and incubate at 37o<\/sup>C for 5 minutes<\/li>\n
  4. Fix cells in 2% PFA in PBS and carry out immunofluorescence<\/a>, as normal<\/li>\n<\/ol>\n

    Notes<\/strong><\/em><\/p>\n

      \n
    • 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<\/li>\n
    • 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%<\/li>\n
    • Mitotracker is provided in 50 \u03bcg tubes and should be stored in the dark at -20o<\/sup>C, indefinitely; it is stable in solution for up to 1 month, though we have not tested its effectiveness following solubilisation and storage<\/li>\n
    • CMXROS is a red dye; hence, any co-staining will require a GFP-tagged protein or FITC-conjugated secondary antibody<\/li>\n
    • To label the mitochondrion of procyclic form T. brucei<\/i>, follow the above protocol, but incubate the cells at 27o<\/sup>C for 30 minutes<\/li>\n<\/ul>\n","protected":false},"excerpt":{"rendered":"

      Mitotracker is incorporated into the mitochondria of live cells and retained following formaldehyde fixation. 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