In the first experiment, cells were then treated with either 0 umol L, 200 umol L, or 800 umol L L arginine in a serum free environment. At two days post treatment, cell proliferation was assessed for one plate of cells, and the media was replenished in the second plate of cells. Cell proliferation was then assessed in the sec ond plate 4 days after the initial Tasocitinib treatment. In the second experiment, cells were treated with 0 umol L, 200 umol L, or 800 umol L L arginine with or without N omega hydroxy nor arginine, a polyamine synthesis inhibitor, in a serum free environment. The media was replenished on day 2 post treatment, and cell proliferation was assessed on day 4 post treatment.

Add itionally, a third experiment examined the role of NO biosynthesis in Inhibitors,Modulators,Libraries endometrial RL95 2 cell proliferation cells were treated with either 0 umol L, 200 umol L, or 800 umol L L arginine with or without 7 Nitroindazole, a NOS inhibitor, in a serum free environment. 7 NI was dissolved in ethanol, and all cells not exposed to 7 NI received an equal amount of ethanol. Cell proliferation was assessed according to procedures pre viously described by Kueng et al.Briefly, cells were washed in Dulbecco0s PBS and fixed in 3% glutaraldehyde for 15 min. Fixed cells were washed three times by submersion in de ionized water and air dried, after which they were stained Inhibitors,Modulators,Libraries with crystal violet for 20 min, followed by three washes with de ionized water. Crystal violet was eluted using 10% glacial acetic acid, and the optical density was measured at 590 nm. All experiments were repeated in dependently three times.

Detection of DNA fragmentation RL95 2 cells were transferred to chamber slides in growth media for a period of 24 h, after which they were serum and L arginine starved for an additional 24 hours in an L arginine free media. Inhibitors,Modulators,Libraries Cells were then treated with either 0 umol L, 200 umol L, or 800 umol L L arginine in a serum free envir onment for 24 hours. Cells were washed with DPBS and fixed in a solution of 4% paraformaldehyde in PBS for 60 min, washed with DPBS, and incubated with a permeabilization solution for 2 min on ice followed by two washes with DPBS. DNA fragmentation was detected by incubating cells with a FITC labeled terminal deoxynucleotidyl transferase dUTP nick end labeling solution at 37 C in a humidified incubator. After 60 min, cells were washed three times with DPBS, the nucleus was counter stained with DAPI, and the slides where covered with a coverslip. TUNEL and DAPI staining were assessed using a Inhibitors,Modulators,Libraries Nikon Eclipse Inhibitors,Modulators,Libraries TE 2000 U fluorescence microscope. Three frames per chamber selleck kinase inhibitor were acquired, and the pro portion of cells that were TUNEL positive was counted. The entire experiment was repeated three independent times.