Wallac wizard 2470 gamma counter

According to the previously reported study, U87MG human glioblastoma cells (DS Pharma Biomedical, Osaka, Japan) and PC3 human prostate carcinoma cells (DS Pharma Biomedical) were used as Integrin ɑ_vβ₃ high- and low-expressing cells, respectively^{29 (link)}. U87MG and PC3 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) or Roswell Park Memorial Institute 1640 (RPMI 1640) containing 10% fetal bovine serum, and 100 U/mL of penicillin and streptomycin in a 5% CO₂ incubator at 37 ºC. These cells were sub-cultured overnight in 12-well plates (4 × 10⁵ cells/1 mL), and then pre-incubated in DMEM (1 mL) with or without c(RGDfK) (final concentration: 10 µM) for 30 min at 37 ºC in a humidified atmosphere containing 5% CO₂. After the pre-incubation, the medium was exchanged with DMEM containing ^99mTc-(Ham-RGD)₂ (74 kBq, 1 mL) and then incubated for 60 min. After the incubation, the cells were washed twice with phosphate-buffered saline (PBS) (1 mL) and then lysed with 1 M NaOH (0.4 mL). The radioactivity in the lysates was measured by a Wallac WIZARD 2470 gamma counter (PerkinElmer, Waltham, MA, USA) and total protein concentrations in samples were measured using the bicinchonic acid method.

The copy numbers of cell-surface IGF1R and ZNRF3 were determined by performing saturation binding experiments using the radiolabelled antibodies against IGF1R (cixutumumab) or ZNRF3 (ZNRF3–55). For saturation experiments, cells were incubated with increasing concentrations of ¹²⁵I-labelled antibodies in binding buffer (OptiMEM, 2% FBS, 50 mM HEPES, and 0.1% sodium azide) for 12 h at room temperature under gentle agitation. Nonspecific binding was determined by pre-incubating cells with an excess of non-labelled antibodies prior to addition of ¹²⁵I-labelled antibodies. Cells and antibodies were transferred to Millipore multiscreen filter plates, washed four times with binding buffer, and allowed to dry. The dried filters were punched into 5 ml polystyrene tubes (Corning) and the radioactivity in counts per minute (CPM) was measured using a Wallac WIZARD 2470 Gamma Counter (Perkin Elmer) for 1 min with 0.8 counting efficiency. Data were fit to a one-site specific binding curve in GraphPad Prism 8.