This was followed by incubation for 45 minutes at 37 C in 50 ml o

This was followed by incubation for 45 minutes at 37 C in 50 ml of Hanks balanced salt solution containing 0. 05% collagenase, with continuous stirring. DNAase in 1. 0 ml of PBS was added 20 to 40 minutes after this incubation period. The cell suspension was fil selleck chemical MEK162 tered, and non parenchymal cells were sepa rated by discontinuous density gradients of Percoll at 1. 044 gml and 1. 07 gml. The final cell suspension was washed twice, and CD133 andor CXCR4 antibody was added and incubated at 4 C for 20 minutes before washing. Stained cells were analyzed using flow cytometry. Inhibitors,Modulators,Libraries The CD133CXCR4 cancer cell content determined by flow cytometry was utilized to investigate the correlation between CD133CXCR4 cancer cells and clinical charac teristics and two year survival.

Suspensions of HCT 116 cells were sorted according to the expression of CD133 and CXCR4 with a fluorescence activated cell sort ing system following multicolor staining as described for flow cyto metric analyses. Inhibitors,Modulators,Libraries Separated subpopulations were reanalyzed for purity and then used in subsequent experiments. Standard tail vein metastatic assay Tumor cells were injected into the lateral tail Inhibitors,Modulators,Libraries vein using a 27 gauge needle, more experimental details were performed as previously described. At 120 days post injection, mice were sacrificed and tissues were examined macroscopically and microscopically Inhibitors,Modulators,Libraries for occur rence of metastases. Clonogenic assay About 5102 cells were added into each well of a six well culture plate. After incubation at 37 C for 14 days, the cells were washed twice with PBS and stained with 0.

1% crystal violet solu tion. The number of colonies containing 20 cells was counted under a microscope. Subcutaneous tumorigenic assay Subcutaneous administration of colon tumor Inhibitors,Modulators,Libraries cells was performed in the armpit area of nude mice. Approxi inhibitor purchase mately 1106 cells were injected at each site. Mice were killed 30 days later, and tumorigenic incidence was assessed. The xenografts were excised for weight evaluation. Real time RT PCR After FACS isolation, cells were cultured in six well plates to 50% to 60% confluence. The treatment group was subjected to SDF 1 at a concentration of 100 ngml for 12 hours. The cells were collected to extract total cellular mRNA with Trizol reagent. Expression of mRNA was determined by real time RT PCR using SYBR Green Master Mix. Total sample RNA was normalized to endogenous GADPH mRNA. The sequences of primers used in this study are shown in Table 1. Thermal cycling conditions included an initial hold period at 95 C for four minutes. this was followed by a two step PCR program of 95 C for 20 seconds and 72 C for 30 seconds repeated for 40 cycles on an Mx4000 system.

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