Rhodamine 123 (Rh123) is cell-membrane permeable and localizes in mitochondria of viable cells to emit yellowish-green fluorescence (Fig. 1). Rh123 is utilized for staining a wide variety of cells, including plant cells and bacteria. Since there is a correlation between the amount of ATP in a cell and the fluorescence intensity of Rh123, this compound is used for the detection of intracellular ATP. Rh123 is also used in cancer research.
Fig. 1 Cell staining mechanism
1. Dissolve 0.4 mg Rh123 in 1 ml DMSO to prepare 1 mM Rh123-DMSO solution.
2. Prepare cells with a glass slide. The cell number will be 5×104 to 5×105 cells per ml.
3. Incubate the slide and wash cells with PBS or Hank’s medium.
4. Dilute the 1 mM Rh123 solution with culture medium to prepare 1-20 μM Rh123 buffer solution.
5. Add the Rh123 buffer solutiona) to the glass slide and incubate at 37oC for 30 min to 1 hour.
6. Remove the Rh123 buffer solution and wash cells with culture medium.b)
7. Observe the cells under a fluorescence microscope with a fluorescein filter.
a) Incubate the Rh123 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. L. V. Johnson, et al., Localization of mitochondria in living cells with rhodamine 123. PNAS. 1980;77:990-994.
2. C. S. Downes, et al., Novobiocin inhibition of DNA excision repair may occur through effects on mitochondrial structure and ATP metabolism, not on repair topoisomerases. Carcinogenesis. 1985;6:1343-1352.
3. G. Varbiro, et al., Direct effect of Taxol on free radical formation and mitochondrial permeability transition. Free Radic Biol Med.