U2OS+SSTR2 cells were seeded in 12-well plates in 1 mL medium and the next day adherent cells (~1×105 cells/ well) were incubated with 5×10-9 M/ 0.25 MBq 177Lu-DOTA-TATE in 1 mL medium for 4 h at 37°C with 5% CO2. Subsequently, cells were washed with PBS. For day 0 measurements, the membrane-bound fraction was separated from the internalized fraction by incubating cells for 10 min in 1 mL 50 mM glycine and 100 mM NaCl, pH2.8. Then, cells were lysed in 1 mL 0.1 M NaOH to collect the internalized fraction. For day 1-6 measurements, medium was added to the cells and they were incubated at 37°C, 5% CO2. For every time point, medium, membrane-bound fractions and internalized fractions were collected as described for day 0. All fractions were counted in a 1480 WIZARD automatic gamma counter (PerkinElmer). Data is expressed as percentage of added dose (%AD). Experiments were performed in triplicate and gamma-counter measurements were corrected for decay.
1480 wizard automatic gamma counter
Manufactured by PerkinElmer
The 1480 WIZARD Automatic Gamma Counter is a laboratory instrument designed for the detection and quantification of gamma-emitting radioisotopes. It provides automated sample handling and analysis capabilities, allowing for efficient and precise measurement of radioactive samples.
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3 protocols using 1480 wizard automatic gamma counter
1
Internalization of 177Lu-DOTA-TATE in U2OS+SSTR2 Cells
2
Quantitative Receptor Binding Assay
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LNCaP-Luc cells were seeded in 24-well plate at a density of 15x10 4 cells per well for 48 h. After removal of growth media, the cells were washed with binding buffer (300 µL, RPMI 1640 Glutamax, 0.5% BSA). 29 (detailed protocol are provided in the ESI) was added to a final concentration of 0.45 nM in the presence of increasing concentrations of ADIBO-KuE conjugates (1 nM-10 µM), PMPA (1 nM-10 µM) or UCNP@KuE (10 pM-100 nM) in binding buffer (final volume of 300 µL). After 1 h of incubation at 37 °C, binding buffer was removed and cells were washed with binding buffer (300 µL). Cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS), washed with PBS (1 mL) and counted using a 1480 WIZARD Automatic Gamma Counter (Perkin Elmer). Half-maximum inhibition constants (IC50) were determined using GraphPad (GraphPad 9.0, San Diego, USA) and are given as mean ± SD.
3
Cellular Uptake of Radiolabeled UCNPs
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For cellular uptake, LNCaP-Luc cells were seeded in 24-well plate at a density of 15x10 4 cells per well for 48 h. Cells were incubated with 18.5 kBq of [ 125 I]UCNP@KuE3 or [ 125 I]UCNP@CO2H in growth medium for 60 min at 37 °C and 5% CO2. After incubation, the medium was quickly removed, the cells were washed with cold PBS, and trypsinized. After centrifugation (450 g, 5 min, 4 °C), pellets were counted for radioactivity using a 1480 WIZARD Automatic Gamma Counter (Perkin Elmer). Results are given as mean of % incubated dose ± SD.
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