Sorted mesothelial cells with or without ischemic button injury in the inner peritoneal wall of model organism Mus musculus
Mus musculus
A modified adhesion induction surgery was done according to previously described procedures on wild type B6(Cg)-Tyrc-2J/J (The Jackson Laboratory). Two ischemic buttons were placed on each side of the peritoneum and no abrasion of the button or abdominal organs was done. Mice were allowed to recover for 6, 12, or 24 hours and euthanized. Ischemic buttons were obtained by cutting the base and placed in dissociation media (DMEM (Life Technologies, 10565-042), 50mg/ml collagenase IV (Worthington Biochemical), 20uM CaCl2) and dissected further using a single edge razor, and incubated in dissociation media for 30 minutes at 37C. The subsequent cell suspension was filtered through a 100um filter and spun and washed with 2% fetal bovine serum (FBS) in PBS. Cells were treated with 1ml of ACK lysis buffer (Life Technologies) for 5 minutes at 4C and spun and washed. Cells were blocked with 1% goat serum (Life Technologies) for 10 minutes and stained with anti-PDPN (BioLegend, 8.1.1, 1:100), anti-LYVE-1 (eBioscience, ALY-7, 1:100), anti-CD31 (eBioscience, 390, 1:100), and anti-CD45 (BioLegend, 30-F11, 1:100) for 30 minutes at 4C. Cells were spun down, filtered, and resuspended in 200ul of 2% FBS in PBS. Cells were sorted using a FACSAria (BD Bioscences); PDPN+LYVE1-CD31-CD45- cells were sorted directly into 750ul of Trizol LS (Life Technologies).