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5?mL PBS, and analyzed by flow cytometry. The three-dimensional tumor spheroids of C26 were established as described previously20?and?21 with some modification. Cells (2��103?per well) were plated onto a 96-well plate pre-coated with 40?��L 2% low melting point agarose. Several days later, spheroids (300�C400?��m diameters) were treated with 0.3?mmol/L CFPE-labeled R8-LP, PEG-LP and CL-R8-LP in the presence or absence of Cys (10?mmol/L) for 4?h. They were rinsed with PBS, followed by fixation of 4% paraformaldehyde for 0.5?h, and the spheroid fluorescent intensities captured by CLSM. In vivo and ex vivo fluorescence imaging experiments were performed Cyclopamine concentration using the Bio-Real in vivo imaging system (Quick View 3000, Bio-Real, AUSTRIA). The tumor-bearing mice were established by subcutaneous inoculation of 1��106 C26 cells in the left flank of BALB/c mice. The C26 tumor-bearing mice were randomly assigned into different groups with three mice each when the tumor diameters reached about 10?mm, and injected intravenously with DID-loaded liposomes at a BML-190 dose of 500?��g DID/kg. Twenty-four hour after injection, mice were imaged with Bio-Real in vivo imaging system, and immediately euthanized with cervical dislocation. Hearts, livers, spleens, lungs, kidneys and tumors were collected. Organs were imaged with Bio-Real in vivo imaging system. DID fluorescence (excitation 644?nm, emission 665?nm) was monitored to localize the liposomes. For the qualitative evaluation of cellular uptake of the DID-loaded liposome in tumors, at 24?h post-injection, Cys (120?mg/kg) or PBS was injected. Tumors were excised and frozen sectioned 4?h later. Nuclei were stained with DAPI and fluorescent intensity of slices was observed via CLSM. Results were expressed as a mean��standard deviation (SD). One-way ANOVA was used to compare differences, significance see more was defined as a P value of