Special double well culture inserts

Wound-healing assays were performed using special double well culture inserts (Ibidi GmbH, Martinsried, Germany). Each insert was placed in a 24-well plate and 3.5 × 10⁴ cells were placed into both wells of each insert with 70 μL of medium containing 2% FBS. At confluence, the culture inserts were gently removed, and cells were fed with fresh DMEM with 2% FBS or DMEM with 2% FBS containing HGF (40 ng/mL), LY294002 (5 µM), or both factors. Each well was photographed at 10× magnification immediately after insert removal for baseline wound measurement (T0), and after 24 h and 48 h with a Nikon DS-Fi1 camera (Nikon Corporation, Tokyo, Japan) coupled with a Zeiss Axiovert optical microscope (Zeiss, Oberkochen, Germany). TO-PRO3 iodide fluorescent dye 642/661 (1:5000 in PBS, Invitrogen, cat. T3605, Carlsbad, CA, USA) was used for nuclei staining. The mean percentage of residual open area compared with the respective open area recovered at T0 was calculated using ImageJ v 1.47 h software. For each experimental condition, four independent experiments were performed in triplicate.

To perform the wound-healing assay, we used special double well culture inserts (Ibidi GmbH, Martinsried, Germany). Each insert was placed in a well of a 24-well plate. Cells were starved for 16 h under serum free conditions, then they were detached, and 3.5 × 10⁴ cells were placed into both wells of each insert with 70 μL of medium containing 2% FBS. When cells are confluent, the culture inserts were gently removed, and cells were fed with 2% FBS DMEM (CTRL), or treated with HGF (40 ng/mL), PF-04217903 (50 μM), HGF+PF-04217903, Src inhibitor-1 (5 μM), HGF+ Src inhibitor-1, all diluted in CTRL medium. Each well was photographed at 10 × magnification immediately after insert removal, for the measurement of the wound (cell-free) area (T0 area considered as 100%), and after 24 h and 48 h with a Nikon DS-Fi1 camera (Nikon Corporation, Tokyo, Japan), coupled with a Zeiss Axiovert optical microscope (Zeiss, Oberkochen, Germany). The mean percentage of residual open area compared with the respective cell-free space taken at T0 was calculated using ImageJ v 1.47 h software. For each experimental condition, four independent experiments were performed in triplicate.

The scratch assay was performed using special double well culture inserts (ibidi GmbH, Martinsried, Germany). Each insert was placed in a well of a 8-well μ-slide (ibidi) and 70 μl of cell suspension (prepared at the concentration of 4.5 × 10⁵ cells/ml) were placed into both well of each insert. At confluence, the culture inserts were gently removed and cells were fed with complete medium. Slides were then exposed to simulated microgravity condition (μg) or kept on ground as control (1g) for 24 hours. Cells were photographed at 10X magnification with a Nikon DS-Fi1 camera (Nikon Corporation, Tokyo, Japan), coupled with a Zeiss Axiovert optical microscope (Zeiss, Oberkochen, Germany) and the mean percentage of residual open area compared to the respective cell-free surface in T₀ using TScratch software^{39 (link)}. Four replicate inserts were used for each experimental conditions and each experiment was performed in triplicate.