The samples were analyzed on a FACS Calibur flow cytometer

ell nuclear antigen, Ki67. The 20 ml reactions were performed in a 96-well plate on an Applied Biosystems 7500HT Sequence Detection System by incubation at 95uC for 10 min, followed by 40 cycles of 95uC for 15 s and 60uC for 1 min. The raw threshold cycle values were analyzed by the 2 method using the spreadsheet 2883-98-9 site developed by Pfaffl to determine normalized expression ratios of target genes. PCR products were also confirmed by electrophoresis on 1% agarose gel. Flow cytometry For each line, cells were grown to 3 different densities in triplicate flasks to determine the S phase proportion by 2 dimensional flow cytometry analysis. For this, cells were washed twice with ice cold PBS and harvested by trypsinization. 18673174 Cell pellets were fixed by adding 5 ml of ice cold 70% ethanol by constant stirring and then left overnight at 20uC. Prior to flow cytometry, fixed cells were washed with PBS. The cell number was adjusted to 106/ml. RNAse was added to cells which were incubated at 37uC for 15 min. This was followed by addition of 200 ml of propidium iodide. Flow cytometry was performed with a Cytomics FC-500 Beckmann Coulter instrument with analysis using FC 500 Beckmann CXP software. Fluorine-18-FluoroDeoxy Glucose uptake MCF-7 cells were pulse labeled with 18F-FDG for 15 min at 37uC and processed as described above for 99mTcDMSA uptake. Effect of Na+ on 99m Tc-DMSA uptake The effect of Na+ on the uptake of 99mTc-DMSA was assessed by incubating cells for 1 h, prior to performing the assay, in two types of media in place of the standard DMEM. The high Na+ medium was composed of 137 mM NaCl, 5.4 mM KCl, 2.8 mM CaCl2, 1.2 mM Mg2SO4, 14 mM Tris and 0.1 mM KH2PO4. The Na+-free medium was similarly composed except that the NaCl was substituted with 137 mM N-methyl-D glucamine. Protein determination Protein content was determined using the Bradford protein assay kit after solubilisation with 1 M NaOH and neutralization with 1 M HCl. Bovine serum albumin was used as standard. thymidine incorporation into DNA MCF-7, MDA-MB-231, pII and PC-3 cell lines grown to different cell densities, were pulse labeled with thymidine for 1 h at 37uC in order to assess DNA synthesis. Cells were washed thrice with ice cold PBS, trypsinized and centrifuged at 10,000 g for 5 min at 4uC. Cell pellets were suspended with ice cold PBS and left on ice for 15 min before centrifugation. Pellets were resuspended in 1 ml of 4% trichloroacetic acid at 4uC and again centrifuged. Washes were repeated in 4% TCA at 4uC until the radioactivity in the supernatant was reduced to background level. To solubilise the DNA, the pellet was resuspended in 0.5 ml of 4% TCA and heated to 90uC for 1 h. Then the cell debris was removed by centrifugation and washed with 0.5 ml of 4% TCA. The resulting supernatants and pellets were pooled, then suspended with Ultima GoldTM scintillation fluid and counted in a Beckman LS 6000 TA liquid scintillation counter. Statistical analysis Charting and statistics were performed using Excel and Graphpad Prismsoftware. All data are expressed as mean 6 standard deviation unless otherwise stated. 22404218 One way analysis of variance was used to determine the statistical differences between groups. Results Uptake of 99m Tc-DMSA RNA extraction and measurement of gene expression Initial experiments were conducted with MCF-7 cells to optimize conditions. Cells were seeded into 25 cm flasks to achieve densities of 30, 50, 70, 80, 90 and 100% confluency. This was done by determini