Brown pre-adipocytes were seeded, grown to confluence and differentiated in 6-well plates as described in section General. Cell starving was performed for 1 h using unsupplemented, glucose-free DMEM. Thirty minutes prior to [18F]FDG incubation, cells were pre-treated with the adrenergic beta-3 agonist (2 μM CL 316,243), adrenergic beta antagonist (2 μM (S)-propranolol), the MCHR1 ligands (20 μM SNAP-7941 or FE@SNAP), or vehicle (baseline). [18F]FDG uptake was performed for 50 min at 37°C (humidified atmosphere, 5% CO2). Supernatant was taken off, cells were washed with ice-cold PBS and finally trypsinized. Radioactive cell fractions were gamma counted (2480 Wizard2, PerkinElmer) and normalized to percentage uptake per well. [18F]FDG uptake under baseline conditions refers to 100% uptake. Three independent experiments were performed in triplicates. Statistical analysis was performed using an unpaired, two-tailed t-test.
2480 wizard2
Manufactured by PerkinElmer
Sourced in United States, Finland
The 2480 Wizard2 is a high-performance gamma counter designed for accurate and reliable measurement of radioactive samples. It features a sensitive detection system and advanced software to provide precise data analysis.
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Itlc sg, by Agilent Technologies (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
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Spark multimode microplate reader, by Tecan (1 mentions)
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Desipramine, by Merck Group (1 mentions)
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1470 wizard gamma counter, by PerkinElmer (1 mentions)
Ph indicator paper, by Merck Group (1 mentions)
Barnstead nanopure purification system, by Thermo Fisher Scientific (1 mentions)
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105 protocols using 2480 wizard2
1
Measuring Adipocyte [18F]FDG Uptake
2
Radiolabeling Trastuzumab with 111In
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DTPA-trastuzumab-IRDye800CW was labeled with 2 MBq of 111In (Covidien, Petten, The Netherlands) per microgram of antibody in 0.5 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 5.4 (2 times the volume of [111In]InCl3 solution) and incubated for 20 min at room temperature under metal free conditions. After incubation, unincorporated 111In was chelated by adding 50 mM ethylenediaminetetraacetic acid (EDTA) solution (to a final concentration of 5 mM).
Radiochemical purity was determined by instant thin-layer chromatography (ITLC) on silica gel strips (Agilent Technologies), using 0.15 M sodium citrate buffer, pH 6.0, as the mobile phase.
The immunoreactive fraction of dual-labeled trastuzumab was determined with BT474 cells as described by Lindmo et al. [18 (link)]. A serial dilution of BT474 cells was incubated with [111In]In-DTPA-trastuzumab-IRDye800CW for 1 h at room temperature. Subsequently, BT474 cells were centrifuged and analyzed in a gamma counter (2480 WIZARD2; PerkinElmer).
Radiochemical purity was determined by instant thin-layer chromatography (ITLC) on silica gel strips (Agilent Technologies), using 0.15 M sodium citrate buffer, pH 6.0, as the mobile phase.
The immunoreactive fraction of dual-labeled trastuzumab was determined with BT474 cells as described by Lindmo et al. [18 (link)]. A serial dilution of BT474 cells was incubated with [111In]In-DTPA-trastuzumab-IRDye800CW for 1 h at room temperature. Subsequently, BT474 cells were centrifuged and analyzed in a gamma counter (2480 WIZARD2; PerkinElmer).
3
Biodistribution of 89Zr-Labeled MSCs in Mice
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C57BL/6 mice were intravenously injected with 8 × 10589Zr‐labeled MSCs. Then, the mice were sacrificed at 2, 8, 24, 72, and 108 h p.i. Samples of major organs and tissues, including the brain, liver, heart, spleen, lung, kidney, stomach, muscle, tibia, femur, and knee were removed and weighed. To isolate the knee joint, an incision was made to reveal the femur, tibia, and knee ligaments. The knee was then carefully excised by transecting the ligamentous and musculotendinous attachments to the distal femur and proximal tibia. The radioactivity of each sample was measured using a γ‐counter (2480 WIZARD2; PerkinElmer, USA), and then the %ID/g per organ was calculated. The biodistribution of the other experimental groups was also evaluated after PET imaging.
4
Quantification of 52Mn Biodistribution
5
Arterial Input Function Measurement
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Arterial blood samples were collected manually at different time points (24 × 5 s, 1 × 60 s, 1 × 120 s, 1 × 300 s, 1 × 600 s, 2 × 1,200 s after injection) from the radial artery. Whole-blood radioactivity concentrations were measured using a γ-counter (2480 Wizard2 automatic γ-counter; PerkinElmer). To obtain the arterial input function (AIF), whole-blood samples were centrifuged to separate the plasma component, followed by the measurement of radioactivity in the plasma. The measured whole-blood and plasma tracer concentrations were used to calculate the time-dependent plasma–to–whole-blood ratios for each subject.
6
Biodistribution of Copper-64 Radiopharmaceuticals
7
Zinc Uptake Assay in Hepa Cells
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Hepa cells were seeded in 24-well plates and incubated for 24 h with DMEM supplemented with 10% FBS in the absence or presence of 1000 μg/mL of P. ginseng extract. The Hepa cells were washed with wash buffer (10 mM HEPES pH 7.4, 142 mM NaCl, 10 mM glucose, 5 mM KCl) and then incubated with 0.5 μM ZnCl2 containing 1.87 nM 65ZnCl2 (18.5 kBq/well) in a zinc-deficient conditioned medium at 37°C for 20 min. Zinc uptake was stopped by adding an equal volume of ice-cold wash buffer containing 1 mM ethylenediaminetetraacetic acid (EDTA). After removing the buffer, the cells were washed two times with the same buffer and dissolved in 200 mM NaOH. Cell-associated radioactivity was measured with an automatic gamma counter (2480 Wizard2, Perkin Elmer, Waltham, MA), and protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). Zinc uptake rates were calculated and normalized to protein content.
8
Radiochemical Stability of [18F]21 and [177Lu]21
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[18F]21 or [177Lu]21 (50 μCi, 10 μL) was added to PBS (90 μL) or murine serum (90 μL) and the mixture was incubated at 37 °C for a certain time ([18F]21 : 2 h, [177Lu]21 : 8 d). The PBS mixture was injected directly into the radio-HPLC for analysis. The murine serum was precipitated with 0.1 mL of acetonitrile and centrifuged (10,000 rpm, 5 min). The supernatant was measured and analyzed by the radio-HPLC to determine the stability. For in vivo stability study, the radio tracers were injected into the tail vein of ICR mice and samples of blood were collected after a certain time post injection (pi) ([18F]21 : 4 h, [177Lu]21 : 9 h). The plasma proteins were precipitated using an equal volume of acetonitrile and centrifuged (10,000 rpm, 5 min). The RCP of radio tracer in supernatants were analyzed and quantified by radio-HPLC. The radio-HPLC method was as follows: A: 0.1% TFA in H2O, B: ACN, 0–10 min, 0–100% B. The flow rate was 1 mL/min, and the C18 column (4.6 × 150 mm, 5 μm, ZORBAX, Agilent) was used. When using HPLC to assess the stability of [177Lu]21 , the effluent was collected every 30 s due to its less radioactivity, and samples were measured by an automatic γ-counter (2480 Wizard2, Perkin Elmer). The counted samples were plotted as intensity (cpm) against fraction [19 (link)].
9
In Vivo Tracking of Adoptive T Cell Therapy
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The experimental setting is based on the tumor mouse model previously described 5 (link). Briefly, 1x107 ML2-B7 and ML2-B15 (control tumor) were inoculated s.c. into the right and left flank of NSG mice, respectively. 7 days after tumor inoculation mice were total body irradiated (TBI) with 1 Gy (Gulmay Irradiation Cabinet) to support the engraftment of the i.v. transferred TCR-transgenic TCM cells. At day 8 after tumor injection different numbers of TCR2.5D6 iRFP TCM or non-transduced TCM as control were injected i.v. in the tail vein. At the day of TCM transfer, mice were additionally injected i.p. with hIL-15 producing NSO cells (previously irradiated with 80 Gy). 3 days later, the radiotracer 89Zr-Df-aTCRmu-F(ab')2 was injected i.v. (2.13 ± 0.17MBq corresponding to 10-20 µg). PET/CT imaging was performed 48 h post 89Zr-Df-aTCRmu-F(ab')2 injection. At the day of imaging the mice were sacrificed, blood and organs were taken, weighed, and measured in the gamma-counter (2480 Wizard2, PerkinElmer) for biodistribution analysis. Tumor and organ uptake were expressed as percentage of injected dose per gram (%ID/g).
10
Evaluating Radioactive Antibody Binding Activity
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According to a previously
described flow cytometry protocol, cellular binding activity of Sn-mAbs
and 211At-mAbs was evaluated 6 days after 211At-labeling.29 (link)Using a previously
described in vitro binding assay with minor modifications,3 (link) we evaluated the binding activity of 211At-mAbs the day after labeling. In brief, serially diluted OE19 or
NUGC-3 cells in PBS containing 0.1% bovine serum albumin (BSA) and
2 mM ethylenediaminetetraacetic acid (EDTA) (BE-PBS) were incubated
for 30 min on ice with 5 kBq of 211At-mAbs. Then, samples
were washed three times with BE-PBS. The radioactivity bound to the
cells was counted using a gamma counter (2480 Wizard2;
PerkinElmer, Waltham, MA, USA), and the percentage of cellular binding
was calculated by dividing the radioactivity bound to the cells by
the initially added radioactivity. The immunoreactivity of 211At-mAbs was determined according to the method of Lindmo et al.34 (link)
described flow cytometry protocol, cellular binding activity of Sn-mAbs
and 211At-mAbs was evaluated 6 days after 211At-labeling.29 (link)Using a previously
described in vitro binding assay with minor modifications,3 (link) we evaluated the binding activity of 211At-mAbs the day after labeling. In brief, serially diluted OE19 or
NUGC-3 cells in PBS containing 0.1% bovine serum albumin (BSA) and
2 mM ethylenediaminetetraacetic acid (EDTA) (BE-PBS) were incubated
for 30 min on ice with 5 kBq of 211At-mAbs. Then, samples
were washed three times with BE-PBS. The radioactivity bound to the
cells was counted using a gamma counter (2480 Wizard2;
PerkinElmer, Waltham, MA, USA), and the percentage of cellular binding
was calculated by dividing the radioactivity bound to the cells by
the initially added radioactivity. The immunoreactivity of 211At-mAbs was determined according to the method of Lindmo et al.34 (link)
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