Flasks containing spheroids with diameters of ∼100 μm were divided into three groups: direct irradiation, bystander, and control. The direct groups were irradiated at room temperature with 6-MV X-rays produced by a linear accelerator (Linac 600, GMV; Varian Medical Systems; USA).
The bystander group received medium from the target group, and the control (sham irradiated and sham bystander) flasks were handled under conditions similar to the target, but without irradiation. Irradiated cell conditioned medium (ICCM) was collected from the target and sham irradiated flasks incubated for 1 h at 37°C after irradiation. The transfer of medium was set up using the transfer technique developed by Mothersill and Seymour [1 (link)]. The ICCM pooled for the target and sham irradiated flasks was filtered through 0.22-μm acetate cellulose filters (Orange Scientific; Belgium) and transferred into bystander flasks. Control and target groups were then transferred into the fresh culture medium and incubated at 37°C.
The bystander group received medium from the target group, and the control (sham irradiated and sham bystander) flasks were handled under conditions similar to the target, but without irradiation. Irradiated cell conditioned medium (ICCM) was collected from the target and sham irradiated flasks incubated for 1 h at 37°C after irradiation. The transfer of medium was set up using the transfer technique developed by Mothersill and Seymour [1 (link)]. The ICCM pooled for the target and sham irradiated flasks was filtered through 0.22-μm acetate cellulose filters (Orange Scientific; Belgium) and transferred into bystander flasks. Control and target groups were then transferred into the fresh culture medium and incubated at 37°C.