Page 120 - Physico-Chemical Niche Conditions for Bone Cells

Regulation of bone cell mitochondrial structure and dynamics
MATERIALS AND METHODS
Cell culture
MC3T3-E1 pre-osteoblasts were cultured in α-minimal essential medium (a-MEM; Gibco, Paisly, UK) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% penicillin (Sigma- Aldrich, St. Louis, MO, USA), 1% streptomycin (Sigma-Aldrich), and 5% fungizone (Gibco) in a humidified atmosphere of 5% CO2 in air at 37°C. After reaching ~ 90% confluency, cells were detached using 0.25% trypsin (Gibco) and 0.1% ethylenediaminetetraacetic acid (EDTA; Merck, Darmstadt, Germany) in phosphate-buffered saline (PBS; Sigma-Aldrich, St. Louis, MO,USA)at37°C,seededat1.5´105 cellsper75cm2 cultureflask(GreinerBio-One, Kremsmuenster, Austria), and cultured until the cell layer reached ~90% confluency again. Medium was changed every 3 days. MC3T3-E1 pre-osteoblasts were used for PFF experiments as described below.
PFF treatment of bone cells and video/image acquisition
One day before PFF-treatment, MC3T3-E1 pre-osteoblasts were harvested and seeded at 1×103 cells/cm2 on poly-L-lysine-coated (50 μg/ml; poly-L-lysine hydrobromide; Sigma) glass slides (24×24×0.15 mm). Prior to 4 h PFF, cells were incubated using a-MEM with 2% FBS containing 100 nM MitoTrackerÒ Mitochondrion-Selective probes (Invitrogen, Fisher Scientific, Carlsbad, CA, USA) for mitochondria or 250 nM SiR-actin (Spirochrome, Stein-am-Rhein, Switzerland) for F-actin at 37°C in the dark. To determine the effect of PFF on mitochondria and F-actin, the glass slide containing the cells was placed in a microfluidic chamber connected to a pump. Cells were treated by PFF as described earlier [35]. They were subjected to PFF for 2 min by pumping 7 ml a-MEM medium with a shear stress of 6.5 Pa/s (peak shear stress rate), 1.0 Pa (amplitude), and 1 Hz (frequency). Leica TCS SP8 confocal microscopy (Leica Microsystems, Wetzlar, Germany) was utilized to take videos of mitochondria and F-actin at both top view and side view. XY and XZ time-lapse videos were acquired using a 40´ 1.3 NA oil objective.
Video/image analysis
Videos/images were analyzed using Image J software (https://imagej.net/Fiji/Downloads). For video analysis of mitochondrial movement, the plugin of Tracking was used [36]. First, the video or image was opened by Image J, transferred RGB Color into 8-bit, and adjusted Brightness/Contrast using Auto click. Second, Tracking was found in the list of Plugins, where TrackMate can be found and opened (Fig. 2A). For image analysis of mitochondrial footprint, network branch, and branch length (Table 1), the plugin of MiNA was used. First, the images
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