Cells were selleck chemical SB203580 plated at a concentration of 2 105 cells/ml until next day when they had reached at approximately 80% confluence. The medium was replaced with serum free one and cells where stimulated with CRF at a concentra tion of 10 8 Inhibitors,Modulators,Libraries M for different time points. Control cells were treated with the CRF diluent, being 0. 1% acetic acid. When antagonists or inhibitors were used, antagonists were administered one hour prior to stimulation with the peptide. a helical CRF and astressin 2B, antagonists of CRF1 and CRF2 respectively, were used at a concentration of 10 6 M. RNA isolation and Reverse Transcription PCR Total cellular RNA was isolated using Trizol reagent. Following reverse transcription, 1l of the cDNA product was amplified by PCR.

The primer sets, previously reported to detect the respective CRF receptors in human brain Inhibitors,Modulators,Libraries specimen Products were amplified using the following PCR conditions Inhibitors,Modulators,Libraries denaturation at 95 C for 45 seconds, annealing at 60 C for 45 seconds, exten sion at 72 C for 45 seconds, for a total of 40 cycles. A sam ple where no reverse transcriptase was added during reverse transcription Inhibitors,Modulators,Libraries of the RNA and another where water was added instead of cDNA, were used as controls. 12l of the amplified products were separated on a 2. 5% agarose gel and visualized by ethidium bro mide staining using the BioRad Molecular Analyst System. Quantitative measurement of apoptosis The APOPercentage apoptosis assay was used to quantify apoptosis, according to manufacturers instructions.

Briefly, cells were Inhibitors,Modulators,Libraries plated in flat bottom 96 well plates at a concentration of 15,000 cells/well and the next day medium was replaced neverless by medium free of serum containing CRF at a concentration of 10 8 M, for different time points. One hour before the end of the experiment, 5l of the APOPercentage dye was added to each well for one hour. Cells were then washed with PBS and lysed in the Dye Release Reagent. The APO Percentage Apoptosis Assays dye stained red the apop totic cells undergoing the membrane flip flop event, when phosphatidylserine is translocated to the outer leaflet. Apoptosis was quantified after cell lysis by measuring the dye incorporated in apoptotic cells at 550 nm using an Elisa reader. Quantitative measurement of cell growth Cell growth was measured using the yellow tetrazolium MTT assay. The yellow tetrazolium MTT is reduced by metabolically active cells, in part by the action of dehydrogenases. Briefly, 15,000 cells were plated in 96 well plates and treated with CRF At the end of the incuba tion period MTT was added at a concentration of 0. 5 mg/ ml and incubated for 3 hours. Cells were then lysed by adding 0. 04 N HCl in isopropanol and absorbance was measured at 620 nm in an ELISA plate reader.