Biostation

Scratch test for registering the capacity of PRGF and PRF compounds to induce wound healing was used the test for monitoring the actual fibroblasts migration on a standard gap (14 (link)). Fibroblasts were seeded at a density of 0.33×10⁶ cells/IBIDI plate and 24 h of cultivation at 37°C in 5% CO₂, the silicon frame was removed displaying a standard gap of 500 µm (Fig. 3). The plates were washed thoroughly with PBS to remove any debris. Fibroblasts in the presence of 3 ml complete medium with/without 10% serum (controls) or in the presence of the tested compounds were incubated and migration registered by video capture in BioStation (Nikon Corporation, Tokyo, Japan).

Long-term imaging was done using a microscope equipped with temperature, humidity, and CO₂ control (Olympus IX81 inverted microscope) or a BioStation (Nikon). Phase contrast images were acquired every 2 min or 30 s using a 10× (NA, 0.3, Ph1) or 20× (NA, 0.45, Ph2) objective. Acquisitions were typically obtained over a period varying from 4 to 24 h. Short-term imaging was performed using an Olympus IX81 inverted microscope with a 20× or 60× differential interference contrast (DIC; NA, 1.2, DIC1, water) with an acquisition every 2 s for a few hours. For high-resolution microscopy, spinning disk confocal microscopy was performed with a Perkin-Elmer confocal spinning unit connected to an IX 81 microscope body (Olympus). Images were acquired through a 60× (NA, 1.2, water) objective with a C9100-13 electron multiplying charge coupled device camera (Hamamatsu Photonics). A confocal LSM70 Observor Z1 (Carl Zeiss) with a 63× (NA, 1.2, water) objective was also used for image acquisition. MetaMorph software (for Olympus microscope; Molecular Devices), Nikon software (for BioStation), and ZEN 2006 software (for Carl Zeiss microscope) were used. For life cell imaging, all images were acquired with cells in high glucose DMEM supplemented with 10% Hi-FBS (Invitrogen) at 37°C with 5% CO₂.