Fig. 3. Kinetics of -H2AX and Rad51 foci formation in Namalwa and WI-L2-NS cells after irradiation. (A,B) Starting from 3 minutes after 10 Gy irradiation, cell samples were taken, cytospun, fixed and stained for -H2AX and the amount of cells expressing -H2AX foci scored by immunofluorescent microscopy. (A) A histogram representing the relative frequencies of -H2AX foci per Namalwa cell, 24 hours after irradiation. The number of -H2AX foci per nucleus appeared to be dose dependent with a median number of foci of 14, 30 and 42 for 5, 10 and 15 Gy, respectively. (B) The proportion of the -H2AX foci-containing cells 24 hours post-irradiation was 100% and remained high for 2 days for Namalwa (open square) and 4 days for WI-L2-NS (black circle) cells. Then it consequently decreased reaching nearly zero for Namalwa on day 7 and 6.5% for WI-L2-NS cells on day 8. Inset: immunoblotting analysis of Namalwa whole cell lysates of -H2AX, 24 hours after irradiation. (C) The abundance of Namalwa (open aquare) and WIL2-NS (black circle) cells with Rad51 foci was detected on days 2 to 6-7 after 10 Gy irradiation. Cell samples were taken, cytospun, fixed and stained for Rad51 and the amount of cells expressing Rad51 foci scored by immunofluorescent microscopy. Insert: immunoblotting for Rad51 expression level. No difference in the overall level of Rad51 protein was found between untreated and irradiated cells. Data represent averages from at least three experiments±s.e.m.