NOD/SCID mice bearing U87 and U87-stb-CXCR4 xenografts were injected intravenously with 0.74 MBq (20 μCi) of [111In]G5-X4 or [111In]G5-Ctrl formulated in 200 μL of sterile PBS. Mice were sacrificed at 1, 3, 24, 48, and 120 h post injection, and blood, tumors, and selected organs were dissected and weighed. The radioactivity in the collected samples was measured using a Perkin Elmer-2480 Automatic Gamma Counter (PerkinElmer, Waltham, MA, USA). In the blocking experiment, mice were injected with 2.33 mg/kg (45 μg) of unmodified POL3026, 60 min prior to intravenous injection of [111In]G5-X4 or [111In]G5-Ctrl. To calculate the percentage of injected dose per gram of tissue (%ID/g), triplicate radioactive standards (10% of the injected dose) were counted along with samples.
2480 automatic gamma counter
Manufactured by PerkinElmer
Sourced in United States
The 2480 automatic gamma counter is a laboratory instrument designed for the detection and measurement of gamma radiation in samples. It provides accurate and reliable quantification of radioactive isotopes in a variety of applications.
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17 protocols using 2480 automatic gamma counter
1
Biodistribution of [111In]G5-X4 in U87 Xenografts
2
Time-Dependent Internalization of Radiolabeled Probe
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Approximately 3.0 × 105 SSTR2highAR42J cells were seeded onto each well of a 6-well plate and maintained for 24 h in a humidified incubator at 37 °C with 5% CO2. After gently washing the cells with the binding buffer (20 mM tris, 150 mM NaCl, pH 7.4), a fixed amount (~5 × 105 CPM) of [64Cu]Cu-DOTA-PEG3-TZ(PEG4-Octr)-PEG2-Trz-PEG3-Val-Cit-pABOC-FTY720 diluted with 400 µL of the binding buffer was added to each well. After incubation of the cells for 3, 10, 30, 60, 90, and 120 min (n = 3 each time point), the cells were thoroughly washed with ice-cold binding buffer for the removal of unbound [64Cu]Cu-DOTA-PEG3-TZ(PEG4-Octr)-PEG2-Trz-PEG3-Val-Cit-pABOC-FTY720. Then, the cells were incubated for 5 min with 0.5 mL of ice-cold stripping buffer (150 mM NaCl, 50 mM glycine, pH 3.0) to further remove surface-bound [64Cu]Cu-DOTA-PEG3-TZ(PEG4-Octr)-PEG2-Trz-PEG3-Val-Cit-pABOC-FTY720. After removal of the stripping buffer, the cells were lysed with 500 µL of 1 M NaOH (15 min at 37 °C). The solutions were collected for radioactivity counting on a PerkinElmer 2480 automatic gamma counter (Richmond, CA, USA) to quantify the internalized percentage (Internalized/Total Bound%) of [64Cu]Cu-DOTA-PEG3-TZ(PEG4-Octr)-PEG2-Trz-PEG3-Val-Cit-pABOC-FTY720. The experiment was repeated in triplicate, and data were represented as the mean ± standard deviation (s.d).
3
PSMA Binding Affinity of DUPA Dendrimers
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A competitive cell-binding assay of G1-(DUPA)4, G3-(DUPA)16, and G5-(DUPA)64 was performed to evaluate PSMA binding affinity. A reported PSMA 125I-radioligand [21 (link)] was used as a PSMA-specific radioligand in the assay. PC3-PIP cells were suspended in Tris-buffered saline (TBS) and seeded on multiwell DV plates (Millipore) with 5 × 104 cells per well. The seeded wells (200 µL) were then incubated at room temperature for 2 h (n = 4) with the 125I-radioligand (33,000 cpm/well) in the presence of increasing concentrations (0−2500 nM) of G1-(DUPA)4, G3-(DUPA)16, and G5-(DUPA)64. The unbound 125I-radioligand was removed by filtration. The wells were rinsed five times with cold TBS buffer. The filters were then collected and their radioactivity was measured on a 2480 automatic gamma counter (PerkinElmer). The best-fit IC50 values were calculated for G1-(DUPA)4, G3-(DUPA)16, and G5-(DUPA)64 by fitting the data with nonlinear regression using GraphPad Prism 6.0.
4
Plasma Fractionation and Imaging
5
Biodistribution of [18F]RAGER in Rodents
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Four animals were utilized per time point (two males (weight: 260 ± 30 g) and two females (weight: 200 ± 15 g) to account for any gender differences), and we reasoned these were sufficient numbers for a preliminary biodistribution study. Each animal was intravenously injected with [18F]RAGER via tail-vein (n = 16, 3.0 ± 1.5 MBq, 80 ± 40 μCi), and at 5, 30, 60, and 120 min post-administration, animals were euthanized and dissected. Each tissue was weighed and analyzed for radioactivity using a PerkinElmer 2480 automatic gamma counter and full details are provided in the Supplementary data. Biodistribution studies confirmed brain permeability and quick washout, with radioligand essentially absent from the brain at the 60 and 120 min time points (Fig. S1 ). Limited exposure was observed in other organs, and some accumulation was observed in the later time points in the small intestines. No bone uptake was observed indicating no evidence of metabolic defluorination of [18F]RAGER.
6
Biodistribution of [177Lu]Lu-DOTA-Miltuximab® and [89Zr]Zr-DFO-Miltuximab® in Mice
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On days 3, 5, 7, and 27 post injection of 6 MBq [177Lu]Lu-DOTA-Miltuximab®, and day 7 post injection of 2.62 MBq [89Zr]Zr-DFO-Miltuximab®, mice were euthanised, and the blood was sampled and tissues were collected, cleaned of excess blood and weighed for ex vivo analysis of tissue radioactivity using a PerkinElmer 2480 Automatic Gamma Counter. The gamma counter was calibrated using known samples of 89Zr or 177Lu as appropriate, and measured activity was presented as percent injected activity per gram %IA/g or %IA/organ with the aid of suitable standards of the injected activity and represented as mean ± SD.
7
Biodistribution of 177Lu-DOTATATE in Mice
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Mice were euthanized 24 hours post-177Lutetium (Lu)-DOTATATE injection (2 MBq/mouse). In addition to the tumors, the following organs were harvested: heart, lungs, liver, spleen, kidneys, stomach, small and large intestine, muscle, and femur. The counts per minute (CPM) readings, obtained using a 2480 automatic gamma counter (Perkin Elmer), were normalized by tumor weight and biodistribution data were presented as the percentage of the injected dose per gram of tissue (%ID/g).
8
Cellular Uptake of 18F-FDG and 18F-FMISO
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The LoVo and HT29 cells were cultured at 37 °C in a hypoxic atmosphere of 1% O2 for 24 h prior to 18F-FMISO uptake or in corresponding serum-free medium at 37 °C atmosphere of 5% CO2 for 12 h prior to 18F-FDG uptake. The cell density was adjusted to 5 × 105 cells/100 µl. For the X group, 100 µl of cell suspension were incubated with the same volume of radiotracer (18F-FDG or 18F-FMISO) and buffer (DMEM containing 0.2% bovine serum albumin) in a glass tube at 37 °C for 30, 60, 120 and 240 min. The mixture of 100 μl of radiotracer and 200 μl of buffer was used as a control group (the O group). A volume of 100 μl of radiotracer was used to measure the radiotracer doses (the T group). After incubation, the O and X groups were centrifuged at 1000 rpm/min for 5 min and the supernatants were removed. The radioactivity was measured using a PerkinElmer 2480 automatic gamma counter (Waltham, MA, USA). The ratio of cellular radionuclide uptake was calculated using the following formula: X(cpm)-O(cpm)/T(cpm)%. cpm: counts per minute. The experiments were independently repeated three times.
9
Biodistribution of 177Lu-DOTATATE in Mice
10
PSMA-Targeting Radioligand Binding Assay
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Cell-binding potency of [18F]F-TZ(PSMA)-LEGU-TLR7 was measured with PSMA+ PC3-PIP and PSMA− PC3-Flu cells. Cells were seeded in 6-well plates (5.0 × 105 cells per well, n = 3) and incubated for 24 h in a humidified incubator at 37 °C with 5% CO2. Afterward, binding buffer (20 mM tris, 150 mM NaCl, pH 7.4) was added to rinse the cells and incubated for 1 h at 37 °C with [18F]F-TZ(PSMA)-LEGU-TLR7 (~7.1 × 105 CPM) in 500 μL binding buffer. For the analysis of non-specific binding, PSMA positive and negative cells in 6-well plates were incubated for 1 h with PSMA-targeting ligand Lys-Urea-Glu (1 mM), followed by the addition of [18F]F-TZ(PSMA)-LEGU-TLR7 (~7.0 × 105 CPM). Cold binding buffer was added to rinse the cells, three times. Cells were incubated with 0.5 mL 1 M NaOH at 37 °C for 15 min. The NaOH-digested cells were collected in glass tubes to measure the radioactivity counts in a 2480 automatic gamma counter (PerkinElmer, Richmond, CA, USA).
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