Isolation and Characterization of Peritoneal Cells

Tissues were separated, minced, and incubated in PBS containing 0.2 U/ml of Liberase TM (Roche) and 20 μg/ml DNase I (Roche) in the presence of calcium and magnesium. After digestion, the suspension was further mechanically disrupted by pipetting and filtered through 70-μm cell strainer. Peritoneal exudate cells were harvested by injecting 8 ml of PBS containing 10% fetal bovine serum and 2 mM EDTA into peritoneal cavity. Single-cell suspensions were preincubated with antibody against CD16/32 to block FcγRII/III receptors and stained on ice for 10 min with antibodies conjugated with fluorochrome. Flow cytometry was performed on an LSRFortessa (BD Biosciences), and aldehyde dehydrogenase activity was determined by ALDEFLUOR (StemCell Technologies).