Cells were then treated with either 0 umol L, 200 umol L, or 800 umol L L arginine in a serum free environment for 24 hours, followed by incubation with 5,50,6,60 tetrachloro 1,10,3,30 tetraethylbenzimidazole carbocyanide iodine for 30 minutes at 37 C. Loss of ��m was determined using fluorescence microscopy and flow cytometry. Immediately, following incubation with JC thenthereby 1, fluorescence microscopy was performed using a 490 nm excitation filter, with an orange emission indicating healthy ��m which is due to a potential dependent aggregation of JC 1 molecules in the mitochondria. In contrast, a loss of ��m results in the monomeric form of JC 1 in the cytosol which produces a green emission. For quantification, Inhibitors,Modulators,Libraries JC 1 labeled cells were harvested using EDTA and analyzed by flow cytometry.

Excitation was achieved with a 488 nm argon laser, and emission fluores cence was measured in the FL 1 and FL 2 channels to determine the proportion of cells with JC 1 Inhibitors,Modulators,Libraries monomers or JC 1 aggregates, respectively. From this analysis, the ratio of cells with JC aggregates compared to cells with JC 1 monomers was determined. Flow cytometry analysis was repeated three independent times, and fluorescence microscopy was performed once to obtain representative images. Reverse transcriptase real time PCR RL95 2 cells were transferred to culture dishes 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. Cells were then treated with either 0 umol L, 200 umol L, or 800 umol L L arginine in a serum free environment.

After 24 hours, cells were washed in cold DPBS, trypsinized, and stored as pellets at ?80 C. Total Inhibitors,Modulators,Libraries RNA was isolated, quantified, and reverse transcribed into cDNA using 500 ng of total RNA. For gene expression analysis, NCBI Primer BLAST was used to design primers for BAX, BCL2, and 18s rRNA. Real time PCR was performed using 0. 5 uL of cDNA, a final concentration of 0. 5 uM of each primer, and SYBR Green I Master Mix. The PCR conditions were the following 5 min at 95 C. 40 cycles of 30 sec at 95 C. 30 sec at the optimal annealing temperature. 30 sec at 72 C. Relative gene ex pression was calculated using the 2 CT method. The entire experiment was repeated three Inhibitors,Modulators,Libraries independent times.

In cell ELISA and Western immunoblot detection of BCL2, BAX, BAD, and p BAD proteins Inhibitors,Modulators,Libraries In a 96 well plate, RL95 2 cells were cultured 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. Cells were then treated with either 0 umol L, 200 umol L, or 800 umol L L arginine in a serum free environment. After 24 hours, cells were selleck catalog fixed with paraformaldehyde. BCL2, BAX, BAD, and p BAD expression was assessed using the Pierce Colormetric In Cell ELISA kit as per the manufacturers instructions.