Wallac 1480 wizard3 gamma counter

Seventeen-week-old male and female C57BL/6 mice were purchased to match the age of PET imaging mice. They were anesthetized in the anesthesia chamber using 3% isoflurane in oxygen and received 0.74–1.11 MBq of [¹⁸F]1 in 100 μL of 5–10% EtOH in saline containing 0.5% sodium ascorbate. The injected mice were returned to their cages and free until the time of the study. At 10, 30, and 60 min after the [¹⁸F]1 administration, each mouse (N = 4/time point) was anesthetized again and euthanized by cervical dislocation followed by cardiac puncture to take a blood sample. Then, the following organs were harvested: the brain, blood, heart, liver, lung, stomach, large intestine, small intestine, spleen, kidney, bone, muscle, liquinal LN, interscapular BAT, and tail. The radioactivity of each organ was counted by a PerkinElmer Wallac 1480 Wizard³ Gamma Counter (Waltham, MA). The whole radioactivity that went into the circulation of the mouse was calculated by subtracting the residual radioactivity in the tail from the injected radioactivity. Therefore, the biodistribution was calculated as an average of the percent injected dose per gram of tissue (%ID/g tissue) by dividing the measured radioactivity by the whole radioactivity that actually went into the body and the weight of the tissue.

On the day after irradiation, the medium was changed to fresh medium containing ¹⁸F-FLT (2.0 mL), with the radioactivity set to 1.0 MBq/flask. The flasks were incubated at 37°C under 20% O₂ and 5.0% CO₂ for 1 h, and then, the medium was removed. The cells were washed with ice-cold PBS (2.0 mL) three times, after which 0.1 mol/L sodium hydroxide solution (2.0 mL, Nacalai Tesque) was added, and the cells were lysed by pipetting thoroughly. The radioactivities of the cell lysate (0.20 mL, n = 3) and ¹⁸F-FLT-containing medium used for the cell uptake study as controls were measured using the Wallac 1480 Wizard 3 gamma counter (Perkin Elmer, MA, USA). In parallel, the protein concentration in the cell lysate was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) and then measuring the absorbance at 562 nm using the SpectraMax M5 plate reader (Molecular Devices, CA, USA); a calibration curve was prepared from albumin standards. The percentage of ¹⁸F-FLT cell uptake was normalized to the protein amount (mg).