A MN2-ENHANCED, RGD-DEPENDENT ADHESION TECHNIQUE FOR ISOLATION OF ADULT-RAT TYPE-II ALVEOLAR EPITHELIAL-CELLS FOR IMMEDIATE FUNCTIONAL-STUDIES()

This report describes a Mn2+-enhanced, RGD-dependent adhesion techniqu e for isolation of adult rat type II cells for immediate functional st udies. Lung cells were dissociated by 30 U/ml porcine pancreatic elast ase and 50 mu g/ml trypsin instilled in the airways. Macrophages were selectively removed by adhesion on purified normal goat IgG-coated pet ri dishes. Type II cells were isolated by adhesion for 45 min, on ProN ectin(R)-F-coated dishes in the presence of 0.5 mM Mn2+. The adherent type II cells were then detached with 0.025% trypsin, 2 mM EDTA in Hep es-buffered saline, pH 7.4. The technique yielded 1.5 to 1.7 x 10(7) ( n = 8) cells from a 150- to 200-8 rat. Greater than 90% of the cells w ere pure type II cells as judged by tannic acid staining and immunosta ining with monoclonal antibody 4AmAb, which recognizes pneumocin, a ty pe II cell marker. The technique reduced the time required for cell is olation from the current 16 to 24 h to 2 to 2.5 h, using commonly avai lable laboratory equipment and reagents. Cells isolated by the procedu re were used to study cell adhesion and spreading on purified extracel lular matrix components in the presence of different divalent cations. Mn2+, Co2+, Ni2+, and Mg2+ enhanced adhesion of freshly isolated type II cells to fibronectin and ProNectin(R)-F, while Ca2+ did not promot e type II cell adhesion on these substrata. RGDS peptide at 1 mg/ml co ncentration inhibited the divalent cation-enhanced cell adhesion.