Computational protocol: Cell-SELEX Aptamer for Highly Specific Radionuclide Molecular Imaging of Glioblastoma In Vivo

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Protocol publication

[…] 200 pmol each of FITC-labeled aptamers and GN were incubated with 5×105 U87-EGFRvIII and U87MG cells dissociated with nonenzymatic dissociation buffer on ice for 40 min, respectively. Samples were washed three times and resuspended in 1×PBS. Fluorescence intensity was determined by counting 10,000 events in a FACScan cytometer (Becton Dickinson, Franklin Lakes, NJ). Experimental data were analyzed with Flowjo7.6.1 software (TreeStar Inc, Ashland, USA). [...] U87-EGFRvIII cells (2×104 cells per well) were plated in 96-well plates, fixed with 4% paraformaldehyde for 15 min, blocked with 3% BSA in PBST (0.01% Tween 20 in 1× PBS, pH 7.4) at 37°C for 40 min, washed with PBST and incubated with various concentrations of biotinylated aptamers at 37°C for 40 min. 50 ul of strepavidin-conjugated horseradish peroxidase (1∶1000 in PBS; Sigma, St. Louis, MO) was added into each well and left to incubate for 30 min at 37°C. 50 ul of TMB solution was added and further incubated for 30 min. The reaction was terminated with 25 ul of 2M H2SO4 and absorbance was measured at 450 nm. Apparent equilibrium dissociation constants (K d) values for each aptamer were determined by nonlinear regression according to the equation: Y = Bmax*X/(Kd+X) using SigmaPlot 12.0. [...] Statistical analysis was performed by One-Way ANOVA for analysis of multiple groups or Independent-Samples T Test for analysis of two groups under SPSS 13.0 program. K d values for each aptamer were calculated by nonlinear regression using SigmaPlot 12.0 software. […]

Pipeline specifications

Software tools FlowJo, SigmaPlot, SPSS
Applications Miscellaneous, Flow cytometry
Organisms Mus musculus
Diseases Brain Neoplasms, Glioblastoma