Giemsa solution

Cell survival yields after exposure to half-field and uniform-field under normoxia and hypoxia were evaluated by means of the clonogenic assay, as reported previously. 9 Cells were counted by using a hemocytometer (Erma, Tokyo, Japan) and plated in T25 flasks. Cells were allowed to adhere overnight before irradiation. Immediately after irradiation, cells under hypoxia were released to normoxia, and were incubated for 10-14 days at 37 °C in a humidified atmosphere of 95% air/5% CO2. Colonies were fixed with methanol and were stained with 2% Giemsa solution (Kanto Chemical Co. Inc.). In the same manner as the DNA damage assay, this experiment was also performed in the presence of AG at a concentration of 100 M. It should be noted that the colonies located in the penumbra regions (-1 x
[cm] 1) shown in Fig. 1(B) were excluded from calculating surviving fraction, that is the ratio of plating efficiency of irradiated group to that of the non-irradiated group.

Cell viability after X-ray irradiation was monitored using both the MTS and the colony formation assays. For the MTS assay, 2500 cells were seeded in each well of 96-well plates. After 24 h, the cells were treated with 1 mM sodium phosphate (pH 6.8)-containing culture medium and 1 mM polyP-containing culture medium. Then the cells were irradiated with X-rays (10 Gy), and their growth was assessed by MTS assay. The MTS assay was performed using CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega Corporation). Absorbance was measured at 490 nm by the Model 680 Microplate Reader (Bio-Rad Laboratories, Inc.). For colony formation assay, the cultured cells were seeded onto a culture flask (25 cm 2 growth area) and incubated for 24 h (2 × 10 5 per flask). The cells were then treated with control and polyP medium overnight. The treated cells were then irradiated with X-rays (10 Gy) and were immediately trypsinized. Then 10 4 -10 5 viable cells were re-plated onto a culture dish (60 mm in diameter) and were cultured in DMEM containing each treatment condition. After 14 days, the grown cells were fixed with methanol and stained with 2% of Giemsa solution (Kanto Chemical Co., Inc., Tokyo, Japan) to count the number of colonies per dish. The number counted was corrected using the plating efficiency of the non-treated cells. The plating efficiency of control cells was 60%.