Xcd v50 camera

The MTT reduction assay was performed according to procedures previously described [104 (link), 105 (link)]. In brief, 3 × 10³ cells in 150 μl of DMEM-GFs medium from cultures grown to near confluency in DMEM+GFs, were plated into wells of a 96 well tissue culture plate (Midwest Scientific, Valley Park, MO, USA). After 0, 1, 3 and 5 days 20 μl of 5 mg/ml MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltrtrazolium bromide (Life Technologies, Carlsbad, CA, USA), were added. The plates were then incubated subsequently for 3.5 hour at 37° C in the absence of light. Microscopic images, were then taken using an Olympus CK2 inverted microscope equipped with a digital XCD-V50 camera (Sony, San Diego, CA, USA), and then150μl of a MTT solvent containing 8mM HCl, 0.2% Nonidet P-40 (NP40, Amresco, Solon, OH) in isopropanol was immediately added to each culture. The 96 well plates were then shaken for 15 min at 100 rpm protected from light at room temperature. The absorbance was determined using a Spectra max Plus 384 multiwell plate reader (Molecular Devices, Sunnyvale, CA, USA) at 590 nm. All cultures were compared to day 0. Experiments were performed in triplicate.

Fibroblasts were plated on the collagen-coated insert of a Petri dish as described above. 2D images at 4x or 10x magnification were acquired through an Olympus CK2 microscope housed in an incubator at 37°C and 5% CO₂. Illumination and acquisition were synchronized using Fire-I software (www.unibrain.com/products/fire-i-software/). A series of JPEG images were acquired every 45 seconds with a XCD-V50 camera (Sony, San Diego, CA). The images were imported into J3D-DIAS4.2 [13 (link), 14 (link), 22 (link)–24 ] and compiled into JDIAS movie format for motion and shape analysis. Cells were outlined at 7.5 minute intervals for 6 hours. Stacked perimeter plots were generated and length, width, perimeter, area and instantaneous velocity calculated as described previously [25 ]. J3D-DIAS4.2 calculates persistence as the ratio of net to total path length at 5 frame intervals and averaged over the total path length.

A wound healing assay was employed that consisted of a two well culture dish in which the wells were separated by an insert, which, when removed, formed a gap (“wound”) (IBIDI, Madison, WI, USA). The assay monitored collective directional migration into the gap between the opposing confluent layers of cells [60 (link)]. Experiments were performed in an incubator at 37° C in 5% CO₂. The assay was performed according to the manufacturer's directions. In brief, 80 μl aliquots, containing 4 × 10⁴ cells were inoculated into each of the two wells and grown for 24 hours to confluency in DMEM+GFs medium. The insert was then removed, leaving a 200 μm gap between the opposing confluent layers of cells. Two ml of fresh medium (DMEM+GFs, DMEM+S,-otherGFs or DMEM-GFs) were then added. Migration into the wound was monitored microscopically through an Olympus CK2 inverted microscope equipped with a digital XCD-V50 camera (Sony, San Diego, CA, USA). Images were obtained at noted time intervals.