Biocoat collagen coated 4 well culture slide

Gonadal fat pads were removed intact and washed once in PBS on ice and then transferred to trypsin solution (0.25% trypsin, 0.1% EDTA, Corning diluted 1:1 in PBS) and placed in shaker water bath incubator at 37°C for 30 minutes, with vigorous shaking by hand every several minutes. Intact adipose depots were then removed with forceps and minced and processed as described above (‘‘remainder SVF’’). Wash media was added to the remaining solution which contained mesothelial cells and cells were washed twice prior to plating. For qPCR studies, cells from individual animals were plated on separate wells of a collagen-coated 12-well plate for 1 hour, washed with PBS three times, and lysed in Trizol (Thermo Fisher). For confocal imaging, mesothelial cells from two mice were plated per well of a BioCoat collagen-coated 4-well culture slide (Corning) for approximately 16 hours prior to fixation with 4% para-formaldehyde. After fixation, cells were permeabilized with Triton X-100, stained with Hoechst, and coverslips mounted with Fluoromount-G (Southern Biotech).