2.4. BNCT In Vitro Efficacy Experiment: Clonogenic Assay

U-87 MG, FaDu, and SAS cells were seeded 5 × 10⁵ cells/well into 6-well cell culture plate with culture medium at 37 °C with 5% CO₂ for 24 h. Cells were treated with 500 or 1000 μg/mL of BPA for 3 h. The medium was removed, and cells were harvested into 1.5 mL tubes using Trypsin-EDTA. Cells were centrifuged, and the supernatant discarded. The BPA containing medium was placed in a 1.5 mL tube, and the cells were resuspended. For neutron irradiation, tubes containing U-87 MG, FaDu, or SAS cells were attached to a plate of an acrylic phantom (Figure 1) and irradiated with a thermal neutron fluence of 2, 3, and 4 × 10¹¹ n/cm², respectively. Neutron irradiation was conducted by DM-BNCT. Details about the accelerator and beam parameters can be found in Lee et al. as well as an overview of the A-BNCT system in Korea [24,25]. To accurately measure the thermal neutron fluence, we used two methods: one was the foil activation method, and the other was the Eu:LiCAF scintillation detector. The foil activation method is well-known for neutron flux measurements; we used Au foil and Cd-covered Au foil to measure the thermal neutron fluence [26], and the Eu:LiCAF scintillator is mainly sensitive to thermal neutrons and has low sensitivity to the epithermal and fast neutron energy ranges [27,28]. After neutron irradiation, the control group and neutron irradiation control group were seeded with 200 cells, the U-87 MG BNCT group was seeded with 400 cells, and for the FaDu and SAS BNCT groups, 1600 cells were seeded in the 60 mm cell culture dish (SPL) and incubated at 37 °C with 5% CO₂ for up to 8 days for SAS, 10 days for FaDu, and 11 days for U-87 MG cells. The appropriate number of seeds and incubation time of U-87 MG, FaDu, and SAS were set through preliminary tests. The appropriate number of colonies for clonogenic assay is 20–150 [29]. The cells were washed twice with DPBS, fixed in 10% neutral-buffered formalin solution (Sigma-Aldrich, St. Louis, MO, USA) for 30 min, and stained with 0.01% crystal violet (Sigma-Aldrich) for 60 min. Colonies of more than 50 cells were counted [29,30].

Preparation of in vitro neutron irradiation. (a) The 1.5 mL tubes containing the cells were attached to a plate of an acrylic phantom; (b) acrylic phantom for neutron irradiation; the cells were located about 5 cm in front of the acrylic phantom.