Leica af6000

Scratches on confluent cell monolayer were made with cell culture tips, and the medium was immediately replaced to remove detached cells. The closure of scratch wounds was monitored by time-lapse microscopy for 24 hours using Leica AF6000 (Leica Microsystems, Heerbrugg, Switzerland). Migration velocity was calculated from the migrated distance per hour and presented as μm/h. In CYR61 blocking experiment, cells were pre-incubated with 5 μg/mL anti-CYR61 antibody or control IgG (Sigma, SAB3701275) before conducting the scratches. Representative data from one of the three independent experiments are shown.

Cells on coverslips were placed on a thermostatted (37 °C) inverted Leica AF6000 videomicroscope (Leica Microsystems, Wetzlar, Germany) with 63×, 1.3 numerical aperture (NA) glycerin-immersion objective. Fluorescence excitation was performed with a 427 ± 10 nm bandpass filter through a double band dichroic mirror (440/520 nm). To avoid displacements between donor and acceptor's fluorescence measurements, we used fast detection filter wheel (respectively with 472 ± 30 nm and 542 ± 27 nm bandpass filters). Images were acquired every two minutes. After 6 min, drugs were added to the cell medium by gentle pipetting.
YFP/CFP ratio for cell by cell data analysis was realized with the ImageJ software as described in [10 (link)]. Curves were obtained by subtracting the average of the baseline values (time points 0 to 6 min) from each time point. Histograms were calculated by subtracting the same average of baseline values from the average of plateau values after EGF stimulation (points at time 12 to 20 min). In both cases, average and standard deviation are calculated and represented. p-values were obtained using Student t-test.

To supplement the results obtained using WISH with intermediate stages, epithelia of the developing lower incisor germs of EGFP positive embryos were dissociated (112 Eda−/− and 109 Eda+/+ mouse embryos were used in total). Incisor regions of the lower jaws were dissected and placed into Hank´s solution. The Hank´s solution was replaced by 1% trypsin solution (Difco Laboratories) at 4°C to dissociate the epithelium from the mesenchyme. Dissociated epithelia were documented in the stop solution (20% FCS in Hank´s solution, Sigma Aldrich) using the inverted fluorescence microscope Leica AF6000 (Leica Microsystems GmbH, Wetzlar, Germany). Shh expression was determined according to the green fluorescence.

Left and right M1 tooth germs of ShhEGFP⁺ mouse embryos at 14.3 dpc were dissected identically from the embryonic lower jaw. In particular, one paid attention that the amount of the mesenchyme was almost identical and it should influence left and right M1 germs in the same way. The left M1 germ was then cut into anterior and posterior parts (for details, see Fig 5). Both parts were cultured separately on PET track–etched membrane according to the method described previously in [84 (link)]. Contralateral intact M1 dissected tooth germ from the same specimen was used as control. Cultures were photographed daily using an inverted fluorescent microscope Leica AF6000 (Leica Microsystems GmbH, Germany), from the day of dissection up to day 6 of culture.

Primary HUVECs were seeded in 12-well plates coated with 1% fibronectin at 150,000 cells per well in HUVEC culture medium. After 24 h, the media were replaced with transfection medium as previously described. Then, transfection was conducted, and a scratch wound was performed across the diameter of each well using a p200 pipette tip. Next, cells were washed with PBS and fresh starving medium, and EBM-2 (Lonza) containing only 0.2% FBS and 1% gentamicin amphotericin of the provided BulletKit was added. In order to monitor scratch-wound closure, live phase-contrast microscopy (Axiovert 40C, Carl Zeiss, Oberkohen, Germany) was used for taking pictures at 0 and 18 h after introducing the scratch wound. In addition, a live cell microscope (Leica AF6000, Leica Microsystems, Tokyo, Japan) was used for taking picture every 4 h after scratch until 20 h in the timeline experiment. Pictures were taken in the same location at two positions in each well. Where necessary, pictures were contrast-enhanced using Microsoft PowerPoint. Scratch size was calculated using the wound healing tool macro for ImageJ. Finally, cells were washed with PBS, and 0.5 mL TRIzol/well was added for RNA isolation. Each single scratch assay condition was performed in triplicate, and the scratch-wound healing assay was performed three independent times.