2.4.2. MitoTracker® Red FM-viability test for cultured cells

1. Let the cells adhere to the glass surface of a chamber slide for 24 h.

2. Centrifuge the chamber slide for 10 min at 220 rcf (Eppendorf 5810 R, rotor A-2-DWP).

3. Aspirate the medium.

4. Add 100 µl of freshly prepared MitoTracker® Red FM (300nM) diluted in BM3 medium (Table 1) supplemented with 250 µg/ml penicillin/streptomycin-   solution (Roth, HP10.1)) and 2.5 % antibiotic/antimycotic-solution (Sigma-  Aldrich, A5955).

5. Incubate for 1 hour at 27°C.

6. Centrifuge the chamber slide again for 10 min at 220 rcf.

7. Remove the medium carefully without scratching the surface.

8. Wash the cells with 1x phosphate buffered saline (1xPBS, Table 1)

9. Centrifuge again at 220 rcf for 10 min.

10. Aspirate the PBS-buffer.

11. Fix the cells in 4 % formalin-solution (Roth, 4980.1) for 20 min at room temperature.

12. Centrifuge the chamber slide again for 10 min at 220 rcf.

13. Aspirate the formalin.

14. Wash the cells with 1xPBS

15. Centrifuge again at 220 rcf for 10 min.

16. Aspirate the PBS-buffer.

17. Stain the nuclei with 250 µl DAPI (4′,6-Diamidin-2-phenylindole, VWR,, 1mg/ ml in 99 % methanol) for 5 min in the dark.

18. Aspirate the DAPI-solution and remove the chamber.

19. Wash the cells with 1xPBS-buffer.

20. Let the slide air dry.

21. Cover the cells with ProLong® Gold antifade reagent (Invitrogen, P36930) and a cover slip to preserve the fluorescent dyes.

22. Visualize viable cells under a fluorescence Microscope using a DAPI-filter or a TexasRed-filter.

MitoTracker probes label active mitochondria of living cells and, therefore, allow the identification of individual living cells amongst a cell population and to visually demonstrate the proportion of living cells within a cell population.