Comparative Studies on the In Vitro: MATERIALS AND METHODS(4)
Treatment of Cells
At approximately 80% confluency after first passage, the cell culture medium was changed to RPMI-1640 supplemented with sodium pyruvate, penicillin/streptomycin, 2% stripped fetal calf serum, with or without 36 nM estradiol-17p, 1 ^M MPA, and 100 ng/ml highly purified porcine relaxin (kindly provided by Dr. David Sherwood, University of Illinois, Urbana). The word ‘‘hormones’’ is used in this paper to include both the steroids (estradiol and MPA) and relaxin. buy asthma inhaler
For long-term dbcAMP treatment, 0.1 mM dbcAMP was added with the hormones continuously for 17 days, and the medium was changed every 2 days. Twenty-four hours prior to each treatment time point, the medium was changed to serum-free RPMI-1640 supplemented with sodium pyruvate and penicillin/streptomycin, with or without hormones, and with or without dbcAMP. For short-term dbcAMP treatment, cells were pretreated with hormones only and were exposed to 0.1 mM dbcAMP at 48 h prior to each sampling time point. At each time point, the medium was collected and the IGFBP-1 present in the culture medium was measured using an RIA kit (Diagnostic Systems Laboratories, Webster, TX). Prolactin present in the culture medium was also measured using an RIA kit. Although no prolactin was detectable in our samples, the antibody provided in the commercial kit was able to detect baboon prolactin in cytosolic extracts of baboon term decidua and also amniotic fluid, both of which have high levels of prolactin.