MitoRed is a cell-membrane-permeable, rhodamine-based dye. It localizes in mitochondria and emits red fluorescence (Fig. 1). The interaction of MitoRed with mitochondria depends on the membrane potential of the mitochondria. Mitochondria can be stained with 20 to 200 nM MitoRed. The excitation and emission wavelengths of MitoRed are 560 nm and 580 nm, respectively.
Fig. 1 Cell staining mechanism
1.Dissolve 50 μg MitoRed (1 tube) in 78 μl of DMSO to prepare 1 mM MitoRed-DMSO solution.
2.Prepare cells with a glass slide. The cell number should be 5x104 to 5x105 cells per ml.
3.Incubate the slide and wash cells with PBS or Hank’s medium.
4.Dilute the 1 mM MitoRed solution with culture medium to prepare 20-200 nM MitoRed buffer solution.
5.Add the MitoRed buffer solution a) to the glass slide and incubate at 37ºC for 30 min to 1 hour.
6.Remove the MitoRed buffer solution and wash cells with culture medium.b)
7.Observe the cells under a fluorescence microscope with a rhodamine filter.
a) Incubate the MitoRed buffer solution at 37oC prior to adding to cells.
b) For fixing after washing cells, add 10% formarin buffer and incubate for 15-20 min, and then wash with PBS.
1) R. Ikeda, et al., Effects of Melanin upon Susceptibility of Cryptococcus to Antifungals. Microbiol Immunol. 2003;47:271-277.
Fig. 2 Cell staining with MitoRed
Cell type: HeLa