Ck2 microscope

After 40 h of incubation at 37 °C, cells were imaged live at 20× objective magnification on an Olympus CK2 microscope. For Hoechst 33342 staining, 1500 cells were seeded onto 96-well half-area black microplates and incubated with H₂O₂ as described before. Cells were fixed with 8% formaline in PBS for 10 min at room temperature and simultaneously stained with Hoechst 33342. After staining, wells were rinsed twice with PBS to remove any remaining dye. Apoptotic cells were observed under a fluorescence microscope at 40× objective magnification (Olympus, Tokyo, Japan).

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.

For wound healing assay, samples were processed as previously described^{40 (link)}. Confluent cells seeded in 6-wells plates were scratched once with a p200 tip with a single vertical movement, washed with PBS and returned to the appropriated medium. At the indicated times post-wound, cells were washed with PBS, fixed with 2% PFA in PBS for 20 min RT and stained with 0.01% Crystal Violet PBS solution for 30 min RT. The samples were washed 3 times in H2O and then let air-dry overnight. Images were acquired basally and at 16 h post-wound with a Samsung J510FN camera on Olympus CK2 microscope with A10PL 10× objective. Wound dimensions were quantified with FIJI^{41 (link)}. For western blot of migrating cells, confluent cells seeded in 6-well plates were scratched with a p1000 tip multiple times to obtain alternating confluent cells and free space areas. After 24 h, when most of the cells were moving through the free spaces, samples were lysed together with control unscratched samples, as described in “Western blotting” section. For immunofluorescence of migrating cells, confluent cells seeded onto poly-l-lysine (Sigma-Aldrich) coated microscope slides in 6-well plates were gently scratched with a p200 tip. After 6 h, scratched samples together with control unscratched samples, were processed as described in “Immunofluorescence” section.

Cell invasivity was assayed with CytoSelect 24-Well Cell Invasion Assay (Cell Biolabs, Inc) following the manufacturer's instruction with minor modifications previously described [62 (link)]. 5 individual fields per insert were acquired with Kodak EasyShare C195 camera on Olympus CK2 microscope with A20PL 20X objective. After imaging, crystal violet stained cells were solubilized and absorbance was red at 540 nm using ELx800 Absorbance Microplate Reader (BioTek).

NHDFs, F-NHDF, HPMFs, F-HPMFs, CC-NHDFs or CC-HPMFs were plated onto the collagen-coated insert and overlayed with a Matrigel/MDA-MB-231 cell suspension as described above. 2D images at 4X and 10X magnification were acquired through an Olympus CK2 microscope housed in an incubator at 37°C and 5% CO₂. For 3D analyses of coalescence with DIC optics, a lid with a glass insert was used to cover the dish. 3D cultures were imaged through a 20x objective using Differential Interference Contrast (DIC) optics on a Zeiss Axioplan 2 microscope with a motor-driven stage synchronized to a Zeiss AxioCam MRc5 IEEE 1394 color CCD camera and an LED light source. The microscope was housed in an incubator at 37°in 5% CO₂. A z-series of optical sections was acquired through the preparation at 10 μm increments and this process repeated every 10 minutes for up to 5 days. The z-series was imported into J3D-DIAS4.2 and saved in movie format for 3D reconstructions, as previously described [13 (link), 14 (link), 22 (link), 23 ].