Page 138 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

Chapter 6
to SSTR2-containing regions was quantified using OptiQuant (Perkin Elmer, Rotterdam, The Netherlands) and expressed in density light units/mm2 (DLU/ mm2). Binding of 10-12 M [111In]In-DOTA-TATE to the tissue slices was set at 100%, and the percentage of binding relative to the unblocked tissue sample was calculated for the (partially) blocked tissue sections. These values were used to determine the IC50 value of unlabeled Albutate-1.
4.4. Cell-uptake and internalization of [111In]In-Albutate-1 and [111In] In-DOTA-TATE
Uptake and internalization of the tracers in adhering U2OS-SSTR2 cells was determined as previously reported [37]. U2OS-SSTR2 cells were cultured in Dulbecco modified Eagle medium (Lonza, Geleen, The Netherlands), supplemented with 10% v/v fetal bovine serum (Biowest, Amsterdam, The Netherlands) and 5 mL of penicillin (5,000 units/mL)/streptomycin (5,000 μg/ mL) (Sigma Aldrich), at 37 °C and 5% CO2. Twenty-four hours after seeding (12- well plates, 8 × 104 cells/well) the cells were incubated with 10−9 M of [111In] In-Albutate-1 or [111In]In-DOTA-TATE in 1 mL of culture medium for 1 h at 37 °C. Cellular uptake was stopped by removing medium from the cells, followed by washing twice with 2 mL phosphate-buffered saline (PBS; Gibco, Carlsbad, USA). By incubating cells for 10 min with an acid solution (50 mM glycine and 100 mM NaCl, pH 2.8) the membrane-bound radiotracer fraction was separated from the intracellular fraction. Then the cells were lysed using 0.1 M NaOH to collect the internalized fraction of radiotracer. Activity levels of both fractions were counted in a γ-counter (1480 WIZARD automatic γ counter; PerkinElmer) using a radionuclide-specific energy window, a counting time of 60 s, and a counting error of 5% or less. Data are expressed as percentage of added activity (%AA). Experiment was performed in triplicate and gamma-counter measurements were corrected for decay.
4.5. In vivo biodistribution
The in vivo experiments were done according to the ARRIVE guidelines [38], and approved by the Animal Welfare Committee of the Erasmus MC (AVD101002017867).
In vivo biodistribution studies were performed for [177Lu]Lu-Albutate-1. Male Balb/c nu/nu mice of 4-5 weeks old (Envigo, Horst, The Netherlands) were
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