Entrations of 5 mmol / L were similar to an intracellular Ren level of 6 thioguanine 168 pmol / mg DNA by HPLC 5.11 update these concentrations with those reported in leukocytes out of patients with Crohn’s disease long-term AZA or 6MP treatment received .6 When the 14C-6 MP was added to cells at 37th The addition of the drug at time 0 was performed on ice. Each reaction was stopped with 1 ml ice-cold phosphate-buffered salt solutions Solution over time, as indicated. The cells were immediately centrifuged at 4 for 4 min at 3000 G and the cell pellets were washed three times with ice-cold PBS. The pellet was resuspended in assay buffer Radioimmunpr Zipitation, pH 7.5 piperazinethansulfons Acid 1, 1% Triton X-100, 1% sodium dodecyl sulfate dexoycholate to 0.1%, 10 mmol / l ethylenediaminetetraacetic resuspended Acid, 1 mmol / l dithiothreitol and sodium vanadate, 1 mmol / L) and in a schchen Szintillationsfl. Scintillation fluid was added to make L Soluble and the samples were performed on a Beckman scintillation hlt Counter gez. At least two mutually independent Independent experiments were performed for each cell line, wherein each experiment performed in Dexrazoxane Totect triplicate. Distinguish the simple diffusion-mediated transport Ladungstr Gertransport dosage was cozy the above protocol at 0 for 0 and 60 min and carried out at 37 for 60 min. Each test was performed at least three times. Competitive inhibition of transport 6 MP was measured in 150 ml of transport volume. Non-labeled 6 MP was added to each reaction to a final concentration of 5 mg / ml.
14C-6 MP were then added to each reaction at a concentration of 0.05 mg / mL. Examples contr Were carried out by the addition of an equal volume of water in place to receive from non-radioactively labeled drug to lower the pH of the volume and consistency. The transport assay was performed as above, at 37 min of 60. Each test was performed at least three times. Determination of transport 6 MP was under conditions of free movement of sodium assay performed in 150 ml volume. The cells in each row were washed three times in a buffer heated to claim 37 or sodiumcontaining HEPES buffer, pH 7.4 buffer without sodium or HEPES, pH 7.2. The cells were then Hrchen was in buffer corresponding to a volume of 150 ml of radioactively 14C and 6 MP to each R Resuspended to a final concentration of 0.05 mg / mL. The cells were incubated at 37 for 60 min and then the reaction was quenched by addition of 1 ml ice-cold PBS stopped. The cells were immediately centrifuged and washed three times with ice-cold PBS. The pellet was resuspended in RIPA buffer and schchen into a Szintillationsfl. Scintillation fluid ALK Signaling Pathway was added to make L Soluble and the samples were analyzed on a scintillation Gez counter just increments. Each test was performed at least three times. Colorimetric cell proliferation methyl thiazolyl tetrazolium assay for determining the Lebensf Ability of the cells after culture with 6 MP the MTT assay is a colorimetric test standard to determine cell proliferation and to Lebensf Conductivity. This test was also used to measure cytotoxicity.22, 23 used the MTT assay performed on the B, D, F, H, J, K and L Cells were cultured at an equal number of 6-well plates with the addition of different concentrations of 6 MP plated out. The cells were cultured with 6 MP 3 and 12 days. The MTT assay was acc the manufacterer made S instructions.