Am. J. Respir. Cell Mol. Biol., Vol 10, No. 4, Apr 1994, 347-354.
A Mn(2+)-enhanced, RGD-dependent adhesion technique for isolation of adult rat type II alveolar epithelial cells for immediate functional studies
JS Lwebuga-Mukasa
Department of Internal Medicine, SUNY at Buffalo School of Medicine, Buffalo General Hospital 14203.
This report describes a Mn(2+)-enhanced, RGD-dependent adhesion technique
for isolation of adult rat type II cells for immediate functional studies.
Lung cells were dissociated by 30 U/ml porcine pancreatic elastase and 50
micrograms/ml trypsin instilled in the airways. Macrophages were
selectively removed by adhesion on purified normal goat IgG-coated petri
dishes. Type II cells were isolated by adhesion for 45 min, on
ProNectin-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
Hepes-buffered saline, pH 7.4. The technique yielded 1.5 to 1.7 x 10(7) (n
= 8) cells from a 150- to 200-g rat. Greater than 90% of the cells were
pure type II cells as judged by tannic acid staining and immunostaining
with monoclonal antibody 4AmAb, which recognizes pneumocin, a type II cell
marker. The technique reduced the time required for cell isolation from the
current 16 to 24 h to 2 to 2.5 h, using commonly available laboratory
equipment and reagents. Cells isolated by the procedure were used to study
cell adhesion and spreading on purified extracellular 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-F, while Ca2+ did not promote type II cell adhesion on these
substrata. RGDS peptide at 1 mg/ml concentration inhibited the divalent
cation-enhanced cell adhesion.
