Axio observer z1m microscope

Cells were fixed with 3.7% formaldehyde in PBS and permeabilized with 1% Triton-X. Cells were then immunostained with rabbit polyclonal cortactin primary antibody (H-191, Santa Cruz Biotechnology, Santa Cruz, CA) and Alexa Fluor 594 goat anti-rabbit secondary antibody (Invitrogen) or phospho-Src (Tyr416) Antibody (2101, Cell Signaling Technology, Danvers, MA) and Alexa Fluor 488 donkey anti-rabbit secondary antibody (Invitrogen). F-actin was stained with Alexa Fluor 488 conjugated phalloidin (Invitrogen), and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Images were taken on a LSM 700 confocal microscope (Zeiss, Germany) or an Axio Observer.Z1m microscope (Zeiss, Germany). The presence of podosomes was determined by co-localization (yellow) of F-actin (green) with cortactin (red), a common marker of podosomes [8 (link)].

Conditioned media from the supernatant of cell-filled sutures and was used for cell migration analysis. Here, ASCs (5×10⁵) were filled into sutures as described before and medium was replaced by culture medium containing 2% FCS after 8 days. Conditioned media was collected after additional 48 hours under standard culture conditions. Next, ASCs (1×10⁴) were seeded into a culture-insert (Ibidi, Martinsried, Germany) in 70 μl culture medium. Cells reached confluence after 24 hours and culture inserts were removed. The medium was replaced by conditioned medium or fresh culture media (2% FCS) for controls. After 30 hours, cells were stained with calcein AM (Invitrogen, Oregon, USA), according to manufacturer's instructions, to visualize living cells. Micrographs were taken with a Axio Observer.Z1m microscope (Zeiss, Göttingen, Germany) and cell migration in the open wound area was quantified using TScratch as previously described [17] (link). Results are expressed as the percentage of open wound area at 30 hours compared to 0 hours.