Modulatory Role for Substance P: MATERIALS AND METHODS(2)

Cell Culture and Experimental Treatments Monodispersed cells were plated at a density of 105 cells/ml of MEM supplemented with 10% fetal bovine serum (FBS; Flow Laboratories Ltd., Irvine, UK) into 24-well plates (Coming, Coming, NY), and cultured at 37°C in a 95% air:5% C02 humid atmosphere. Medium was replaced after 48 h of culture with fresh MEM-FBS. After an additional 48-h period, cells were equilibrated with MEM without FBS for 4 h. Thereafter, each well received 1 ml of MEM alone (control) or 1 ml of medium containing test substances at the concentrations specified, and was incubated for 4 h. Test agents included GnRH (Serono, Barcelona, Spain), SP (Sigma), and the GnRH antagonist antide (Sigma). After incubation, medium was collected, centrifuged in a microfuge at 12 500 X g to remove any cellular debris, and kept at -20°C until assayed for porcine (p) LH secretion by enzyme immunoassay (EIA). In order to evaluate the pLH remaining in the cells after treatment, the cell layer was solubilized by adding to each well 1 ml of a buffer consisting of 137 mM NaCl, 8 mM sodium phosphate (pH 7.4), 1 mM PMSF, 1% (v:v) Triton X-100, 0.5% (w:v) sodium deoxycholate, and 0.1% SDS (all purchased from Sigma). After 15 min of incubation under continuous agitation in an orbital shaker, the cell lysate was centrifuged, and the liquid extract was stored at -20°C until analyzed for pLH content by EIA. buy asthma inhaler
Results presented in this study correspond to three independent experiments. Each experiment was carried out separately using 4-6 pooled pituitary glands. A minimum of 6 replicate wells per experiment were tested for each agent. To avoid variability between experiments, samples of pLH secretion or intracellular content from the same experiment were analyzed within the same assay and expressed as a percentage of the corresponding control value. Data are shown as the mean ± SEM of the three experiments.


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