Icap6000 emission spectrometer

For the in vitro ¹⁰B uptake study, B16 mouse melanoma cells, C6 rat glioma cells, and F98 rat glioma cells were used. B16 and C6 cells were used for in vitro testing at Osaka Prefecture University. Based on results from initial in vitro screening, the in vitro ¹⁰B uptake study using F98 glioma cells were performed at Osaka Medical College. Four hundred thousand cells were seeded in a tissue culture dish (100 × 20 mm; Becton Dickinson, Franklin Lakes, NJ, USA) with DMEM supplemented with 10% FBS and 10% penicillin and streptomycin at 37 °C in a 5% CO₂ atmosphere. After incubation for 4 days at 37 °C, the medium was removed, and DMEM with 10% FBS containing 1 mM KA-BSH, BSH, or BPA was added to each dish. The cells were incubated for an additional 24 h at 37 °C. The medium was then removed, and the cells were washed twice with 4 °C phosphate-buffered saline (PBS) and detached with trypsin–ethylenediamine tetraacetic acid solution. PBS was then added, and cells were digested overnight with 1 N nitric acid solution (Wako Pure Chemical Industries, Osaka, Japan), and ¹⁰B uptake was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) using an iCAP6000 emission spectrometer (Hitachi High-Technologies, Tokyo, Japan).

HGG13, HGG30, and TS cells were incubated with 5-aminolevulinic acid (ALA) (Cosmo Bio, Tokyo, Japan)-containing medium (0, 33, 100, 300, and 900 μΜ) for 24 h. After incubation, the cells were centrifuged and washed with phosphate-buffered saline (PBS). Cells were then exposed to 1 mM BPA-containing medium for 6 h, following which the cells were centrifuged and washed twice with PBS. The cells were then digested for 24 h with 1 N nitric acid solution (Wako Pure Chemical Industries, Osaka, Japan). The intracellular concentration of ¹⁰B was measured using inductively coupled plasma atomic emission spectroscopy (ICP-AES; iCAP6000 emission spectrometer, Hitachi High-Technologies, Tokyo, Japan). All the above experiments were performed under dark conditions to avoid the photodynamic effect on ALA-exposed tumor cells.