Cam xm10

Cells were cultivated in DMEM supplemented with 10% fetal calf serum (FCS) (Sigma-Aldrich, St. Louis, MO, USA), 2 mM L-glutamine, 50 U/mL penicillin, and 100 µg/mL streptomycin ((all Lonza, Basel, Switzerland). The cells were transfected using Turbofect (Thermo-Scientific, Waltham, MA, USA) according to the manufacturer’s instructions using 1 µg DNA and 1.25 µg Turbofect. Then, 40 h after transfection, the cells were fixed for 15 min with 4% paraformaldehyde in PBS (phosphate buffered saline). Cells were washed, permeabilized (5 min with 0.1% Triton X-100 in PBS) and blocked in blocking solution (PBS with 5% FCS and 0.5% bovine serum albumin (BSA, Roche, Basel, Switzerland)). After incubation with primary antibodies (rabbit: α-PMP70; 1:2000, ABR, Golden CO, USA), the slides were washed with PBS several times and exposed to compatible secondary antibodies (Cy3-labelled donkey-α-rabbit IgG, 1:400, Jackson Immuno Research Laboratories, West Grove, PA, USA). Finally, the cells were mounted in PBS/glycerol (1:9) with 3% DABCO (Sigma, St. Louis, MO, USA). For microscopic analysis, an inverted microscope IX71 (Olympus, Wien, Austria) equipped with a CCD camera (CAM-XM10) and appropriate filter sets were used together with C-M-cell software (Olympus, Wien, Austria).

Cells were seeded onto 24 × 24 mm glass coverslips and incubated under the indicated growth conditions until they were fixed for 15 min using 3.7% paraformaldehyde in phosphate-buffered saline (PBS), followed by 0.1% Triton X-100 for permeabilization for 5 min, and then blocked in blocking solution (PBS with 10% FCS and 5% bovine serum albumin, Roche Applied Science, Penzberg, Germany) for 2 h at room temperature. Cells were incubated with primary antibodies from different species overnight at 4 °C under a humidified atmosphere. Immunostained coverslips were then incubated with the corresponding secondary antibody for 1 h at room temperature, counterstained by DAPI (4′,6-diamidino-2-phenylindole, Roche Diagnostics Gmbh, Mannheim, Germany) to visualize nuclei, and mounted on glass slides with Mowiol (Sigma, St. Louis, MI, USA)-based mounting medium. Coverslips were rinsed in PBS between each step. Slides were later analyzed with an Olympus invert microscope IX71 equipped with a CCD camera (CAM-XM10) using Cell^M/Cell^R software (version 3.2) (Olympus, Shinjuku, Japan) or a confocal laser scan microscope (Leica SP5, Leica, Mannheim, Germany) using Leica confocal LAS AF software (version 3.3.10134). During the analysis of subcellular distribution, cells showing apoptosis or extremely high expression levels were avoided.

The treated and untreated cells were washed with, and resuspended in, phosphate buffer. The cell pellets were resuspended in 50 nM MitoTracker Red (Thermo Fisher Scientific, Waltham, MA, USA) staining solution in 0.005% DMSO preheated to 29 °C. The cells were incubated for 30 min at 29 °C. After staining, the cells were washed with, and resuspended in, phosphate buffer. Just before imaging, the cells were fixed with 0.1% (w/v) poly-L-lysine solution (Sigma-Aldrich). The cells were imaged with an IX83 inverted microscope (Olympus, Tokyo, Japan) equipped with a Planapochromate 60×/1.42 oil objective and CAM-XM10 cooled CCD digital camera. For visualization of mitochondria using MitoTracker Red, a U-FMCHE filter cube (with excitation spectrum 565–585 nm and emission spectrum 600–690 nm) was used. For control of the cell morphology, the cells were imaged in a bright field. For capturing the images, the CellSens Dimension software (Olympus) was used. One hundred cells per sample were observed in each experiment. The relative fluorescence intensity was measured using ImageJ software and determined as the average of the ratio of raw integrated density and area for 100 cells per experiment normalized to the untreated cells.