Overview of the experimental design used in the co-culture assay.

In each experiment, LCLs of the same genotype were seeded into 6-well plates (cells exposed to direct irradiation) and insert plates (bystander cells). Cells in 6-well plates (indicated by white circles) were irradiated with X-ray at doses of 0, 0.01, 0.1, 1, or 2 Gy. Immediately after irradiation, inserts with a permeable filter and containing bystander cells (filled black circles) were placed on top of the cells in the 6-well plates. After 1 or 20 hours co-culture, the directly irradiated and bystander cells were separated and incubated for an additional 24 hours before CA assay and 43 hours before cell viability assay were performed. Total post-irradiation time was 44 or 63 hours in the cell viability assay and 25 or 44 hours in the CA assay.