Materials and Reagents

Murine Embrionic Fbroblasts (MEF)
Phenol-red free HBSS (Gibco^®, catalog number: 14175-079 )
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Gibco^®, catalog number: 15630-080 )
Tetramethylrhodamine methyl ester (TMRM) (Life Technologies, catalog number: T668 )
Cyclosporin H (Enzo Life Sciences, catalog number: ALX-380-286-M001 )
Hoechst 33342
Carbonylcyanide-p-trifluoromethoxyphenyl hydrazone FCCP (Sigma-Aldrich, catalog number: C2920-10MG )
Trypsin (451) - Trypsin, 0.05% (1X) with 0.53 mM EDTA 4Na, liquid, 20 x 100 ml
(Gibco^®, catalog number: 25300096 )

Equipment

IN Cell Analyzer 1000 (General Electric Company)
37 °C 5% CO₂ Cell culture incubator
12 well pates (Corning, Costar^®, catalog number: CLS 3513 )
FACScan (BD)
Centrifuge spinning speed: 15,682,186 x g (G-force) (13,000 rpm, 8,3 cm radius)

Software

IN Cell Investigator Analysis software (General Electric Company)
BD CellQuest software
FlowJo software

Procedure

For quantitative real-time analysis
1. Cells are plated at 50% cell confluence.
2. Cells are incubated for 30 min at 37 °C in phenol-red free HBSS with 10 mM HEPES, 20 nM TMRM, 2 μM cyclosporine-H, inhibitor of multidrug resistance pump activity but not of the permeability transition pore (multi drug resistance pump activity can affect the mitochondrial lloading with TMRM) and 2 μg/ml Hoechst 33342, nuclear dye that can be used in living cells.
3. Sequential images were taken before (3 time points) and after 4 μM FCCP (3 time points) was injected in a motorized way for TMRM (535 nm excitation filter; 600 nm emission) and Hoechst 33342 (360 nm excitation filter; 460 nm emission) in different fields (4 fields acquired for each well and 6 wells for each experimental condition) every 3 min with an IN CELL Analyzer 1000.
4. Images are automatically analyzed with IN Cell Investigator Analysis software to measure the TMRM intensity. Fluorescence intensity is measured on the average of 10 points randomly selected for each field at the first and the last time point, the background corresponding to an area without cell is removed for each field.
5. The fluorescence intensity measured at the last time point (after applying FCCP) is normalized by the fluorescence intensity measured at the first time point of the experiment.
For FACS analysis
1. Cells are plated at 50% confluence.
2. Cells are trypsinized (at least 2 millions of cells).
3. Cells are centrifuged for 5 min at G-force 13,62.
4. Cells are washed in PBS, resuspended in phenol-red free HBSS with 10 mM HEPES and counted.
5. Cells (300,000 cells per tube, each condition done in triplicate) are resuspended the in phenol-red free HBSS with 10 mM HEPES and 20 nM TMRM in the presence of 2 μM of the multidrug resistance pump inhibitor cyclosporine-H and incubated for 30 min at 37 °C. In parallel, cells are incubated 5 min in the same conditions with an uncoupling agent, 4 μM FCCP, to measure the specific mitochondria staining.
6. FACS analysis for TMRM was performed on BD FACScan with CellQuest software and analysed using FlowJo software. Fluorescence intensity of TMRM was calculated by gating on live cells.
7. The intensity of the fluorescence with FCCP was normalized by the intensity without FCCP in order to rule out the possibility that the difference of the intensity is only or mainly caused by the cell sizes.

Acknowledgments

We thank all the co-authors of the article: Chiaravalli, M., Mannella, V., Ulisse, V., Quilici, G., Pema, M., Song, X. W., Xu, H., Mari, S., Qian, F., Pei, Y. and Musco, G., the other members of the lab Boletta, Casari, G. and Cassina, L. and the San Raffaele microscopy facility (Alembic).

References

Rowe, I., Chiaravalli, M., Mannella, V., Ulisse, V., Quilici, G., Pema, M., Song, X. W., Xu, H., Mari, S., Qian, F., Pei, Y., Musco, G. and Boletta, A. (2013). Defective glucose metabolism in polycystic kidney disease identifies a new therapeutic strategy. Nat Med 19(4): 488-493.