Wizard 3 2480 automatic gamma counter

The osteoblasts in 35-mm dishes (three plates for each group) were incubated in 100 µL of 0.9% saline diluted ^99mTc-MDP (3.7 mBq) for exact 40 minutes at 37°C and 5% CO₂ atmosphere. The medium was aspirated, and then the dish was twice washed gently with 0.45 mL ice-cold 0.9% saline. The cells in each dish were digested with 0.2 mL 1X trypsin-EDTA for 15 minutes. The suspension of cell clusters was pipetted cautiously. The dishes were further washed twice with 0.9 mL of 0.9% saline. The cell suspension was combined with the washing saline. The residual mineral component in dish was washed with 2 mL 100 mmol·L⁻¹ HCl for 15 minutes and the solution was collected. Planar gamma camera images were acquired for the dishes before and after aspirating the medium, and also following the cell removal and acid washing steps, respectively. A ^99mTc standard was measured simultaneously with the samples. The percentage of ^99mTc radioactivity in the cell culture (the cells and the cell-excreted mineral) was calculated based on the gamma imaging data. The ^99mTc radioactivity in the collected medium, digested cell suspension, and HCl samples were also measured together with a ^99mTc standard using a gamma counter (Perkin Elmer “Wizard 3” 2480 Automatic Gamma Counter). The percentage of ^99mTc-MDP in cells and residual mineral fraction was calculated after decay time adjustment.