Lsm 780 scope

The 3D protocol described in [18 ] was used. Briefly, 1 × 10⁴ cells were plated on 12 mm circular cover glasses coated with Matrigel Matrix Growth Factor Reduced (MMGFR) (BD Biosciences) with serum free Mammary Epithelial Cell Growth Medium (MEGM™) media (Lonza, Walkersville, MD) containing the BulletKit™ growth supplement (BPE, hydrocortisone, GA-1000, Insulin). An overlay of MEGM growth media containing 2% MMGFR was added 1 hr after seeding. Spheroids were allowed to form overnight before addition of 100 ng recombinant HGF (R&D Biosystems) or pro-HGF conditioned media for 24 hrs after which cells were stained with 5 uM Cell Tracker Orange (Invitrogen) for 45 min and Hoechst 33342 (Life Technologies Carlsbad, CA) for 5 min prior to imaging. Confocal images were acquired on the Zeiss LSM 780 scope at the Microscopy Imaging and Cytometry Resources Core at Wayne State University School of Medicine.

SUM149 cells were fixed with 10% neutral-buffered zinc formalin (Z-fix) (Anatech, Battle Creek, MI) for 20 min, blocked in 5% BSA with 0.1% Triton X-100 for 1 hr, and incubated with rabbit anti-c-Met (Cell Signaling Technology, Beverly, MA) and mouse monoclonal anti-matriptase M24 antibody [13 (link)] overnight at 4^°C. The following day, cells were washed with PBS, and incubated with anti-rabbit 546 or anti-mouse 488 antibodies (Invitrogen, Carlsbad, CA) in blocking solution for 2 hrs at RT. Cells were washed with PBS and mounted with Prolong gold with DAPI (Invitrogen). Confocal images were acquired on the Zeiss LSM 780 scope at the Microscopy Imaging and Cytometry Resources Core at Wayne State University School of Medicine. Images were analyzed using Volocity^™ software.