Wang et al. BMC Cancer 2010, 10:661 http://www.biomedcentral.com/1471-2407/10/661 Table 2 The irradiation periods of various groups U87 U251 SHG44 Group A (min) 64 64 64 Group B (min) 0.29 0.29 0.29 Group C (min) 0.57 0.57 0.57 Group D (min) 27 25 28 Group E (min) 54 50 56 Group F (min) 27 25 28 Group G (min) 54 50 56 Group H (min) 32 32 32 Page 3 of 9 Group I (min) 64 64 64 wells of 96-well microplates (Nunc, Roskilde, Denmark) and incubated for 2, 4, or 6 days. The culture medium was removed and replaced with 100 μl aliquots of fresh medium, without serum, containing 0.5 mg/ml MTT, followed by 4 h of incubation at 37°C. Next, medium was aspirated from the wells and 100 μl dimethyl sulfox- ide (DMSO) was added to each well, to dissolve forma- zan crystals. Optical density (OD) was measured using an enzyme-linked immunosorbent method. Cell num- bers were derived from OD values by reference to a standard cell number-OD curve. Hoechst staining At 12 h, 24 h, 36 h, and 48 h after treatment with BPA- BNCT, cells were fixed for 10 min in 4% (v/v) parafor- maldehyde, and then incubated with Hoechst 33342 dye (Sigma) (10 μg/ml) for 10 min. After washed with PBS, cells were observed using an inverted fluorescence microscope (IX70; Olympus, Tokyo, Japan). Electron microscopy Forty-eight hours after irradiation, cells were detached from plates, washed, suspended in PBS, and concen- trated by centrifugation. Cell samples were fixed in 2.5% (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), postfixed in 2% (w/v) buffered osmium tetroxide for 2 h, and dehydrated in ethanol. Specimens for transmission electron microscopy were embedded in Epon. Thin sec- tions were cut using an ultramicrotome and double- stained with uranyl acetate and lead citrate. Electron micrography was performed (JEM-2000EX; JEOL, Tokyo, Japan) using an operating voltage of 80 kV. AnnexinV/PI analysis Twelve hours, 24 h, 36 h, and 48 h after irradiation, cells were trypsinized, counted, washed twice in ice-cold PBS solution, and resuspended in 1×binding buffer (10 mM HEPES/NaOH [pH 7.4], 140 mM NaCl, and 2.5 mM CaCl2). Next, 5 μl of annexin V-FITC (AV) (R&D Table 3 The dose components of BNCT 4 Gy Systems Europe Ltd., Abingdon, UK) and 10 μl of propi- dium iodide (PI) (Sigma) were added to 100 μl of cell suspension, followed by incubation for 15 min at room temperature in the dark. Finally, 400 μl of binding buffer was added to each sample, which was held on ice prior to analysis on a FACSCalibur (Becton Dickinson Lab- ware, Franklin Lakes, NJ) flow cytometer. Ten thousand cells per sample were analyzed. Clonogenic assay After irradiation, 200 treated cells were plated in six- well flat-bottomed microplates (Nunc) and incubated at 37°C in a humidified incubator under 5% (v/v) CO2 for 2 weeks. The cells were fixed in methanol and stained with Giemsa’s solution. Colonies ≥ 50 μm in diameter were counted [19]. Western blot analysis Twelve hours and 24 h after irradiation, cells were washed twice in ice-cold PBS and protein extracts of U87, U251, and SHG44 cells were prepared by lysis in RIPA buffer (150 mM NaCl, 1% [v/v] NP-40, 0.5% [w/v] sodium deoxycholate, 0.1% [w/v] sodium dodecyl sulfate (SDS), 50 mM Tris HCl [pH 8], 10 mM EDTA, and 1 mM PMSF [Sigma]) for 30 min at 4°C. Samples were next centrifuged for 15 min at 15,000 g. Protein concen- trations of supernatants were determined. For each sample, 60 μg of protein was loaded on a 12.5% (w/v) SDS-polyacrylamide gel, electrophoresed, and trans- ferred to a nitrocellulose membrane (Protran; Schleicher and Schuell, Florham Park, NJ). Each membrane was blocked for 1 h at room temperature with blocking buf- fer (TBS containing 0.1% [v/v] Tween 20 [Sigma] and 5% [w/v] milk powder). Primary antibodies (applied for 1 h at room temperature, or overnight at 4°C) were: anti-Bcl-2 (Santa Cruz Biotechnology, Inc., Heidelberg, Germany), anti-Bax (Santa Cruz), and anti-actin (mouse monoclonal C-2, Santa Cruz). Antibodies were diluted 1:1,000, except for the anti-actin antibody (1:500). Thermal neutron (Gy) Epithermal neutron (Gy) Fast neutron (Gy) U87 U251 SHG44 0.028 0.026 0.029 0.007 0.007 0.007 2.889 2.675 2.996 g-ray (Gy) 0.423 0.392 0.439 10B(n,a)7Li (Gy) 0.653 0.900 0.529