Modulation of 72-Kilodalton Type IV Collagenase: MATERIALS AND METHODS(1)
WISH cells, a line of human amnion cells (American Type Culture Collection, Rockville, MD; ATCC CCL5) were cultured in 25-cm2 flasks. Cells were grown in Dul-becco’s Modified Eagle’s medium, supplemented with 10% fetal calf serum (Gibco BRL, Gaithersburg, MD), 25 mM Hepes, 110 mg/L sodium pyruvate, and 20 mM L-gluta-mine, in a 95% air:5% CO2 incubator at 37°C with humid atmosphere. flovent inhaler
Vitamin C Stimulation
To analyze the response to ascorbic acid, serum-free medium supplemented with 0.2% lactalbumin hydrolyzate was added to confluent cells and maintained during 24 h. Fresh serum-free media and ascorbic acid in a range from 10 to 200 ^g/ml were added to cells, and incubation was maintained for 4-24 additional hours. In each experiment, control flasks without and with vitamin C (29 ^g/ml, the physiologic concentration in amniotic fluid ) were included. Equimolar amounts of glutathione were included in some flasks to control for the effect of change in the redox potential. After incubation, the cells and the culture media were assayed according to the following techniques.