Costar cell scraper

Seven days after NT injection (unless time was a variable), and 24 h after the final vehicle, BAPN and/or steroid injection, animals were killed by exposure to increasing CO₂ concentrations followed by cervical dislocation. Abdominal wall peritoneal mesothelial cells were collected by removing the skin and pinning out the lateral abdominal wall between the hindlimb and ribcage onto clean foil (S2 Fig). A 1 cm tall section cut from the top of sterile 50 ml Falcon tube (VWR, Lutterworth, Leicestershire, UK) was placed on the exposed mesothelial surface and held down firmly. 0.7ml RNA lysis buffer (RNEasy, Qiagen) was placed inside the ring and the mesothelial surface was scraped for 10–15 seconds using a 1.8 cm wide Costar® cell scraper (Corning). The resulting lysate was removed by pipette and stored at -80°C until required for RNA extraction. Evidence that this method removed only the mesothelial cells was obtained by observing cytokeratin staining of tissue that had and had not undergone this treatment (S3 Fig)

To analyze matrix components, nonadherent HPM control and transfected cells in T75 flasks were first counted and removed by pipetting. Matrix adherent to flask surfaces was washed twice for 5 min with cold hypotonic (1:10) PBS with protease inhibitors to lyse adherent cells. Two hundred μl of PBS was then added and matrix removed with a Costar cell scraper (#3010; Corning Inc, Corning, NY). The matrix suspension was centrifuged at 2400 RPM x 5 min, decanted and 100 μl of 1x PBS and 100 μl of 2x sample buffer (SB) were added. Matrix suspension was kept at -80°C until ready for Western blotting. Lysates from non-adherent cells for Western blotting were prepared from 1 x 10⁶ cells as described [18 (link)]. Twenty μl of each cell lysate which is equal to 1.25 x 10⁵ cells and 25 ul of matrix suspension was then loaded on to 4–12% polyacrylamide gels. Proteins were separated by electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked for 60 min using Odyssey blocking buffer (LICOR Biosciences, Lincoln, NE) and incubated overnight with the primary antibodies. After 60 min incubation with the IR-dye (680RD or 800CW)-labeled secondary antibodies (1:20,000), immunoreactive proteins were visualized using an Odyssey imager (LI-COR Biosciences).