X rad 320 cabinet x ray irradiator

RG2 cells cultured under sterile conditions were treated with 750 nM of VE822-loaded PEG-poly(PDL-co-DO) NPs or free VE822. The cells without treatment were used as control. After treatment for 24 h, the cells were irradiated with 5 Gy using an XRAD 320 Cabinet X-ray irradiator (Precision Xray, CT, USA). Cells were harvested 60 min after radiation and prepared for Western Blot analysis using mouse monoclonal anti-Chk1 (2G1D5, Cell Signaling), rabbit monoclonal anti-pChk1 (SER345) (133D3, Cell Signaling), and Vinculin (Santa Cruz Biotechnology). as a loading control. This protocol was repeated for non-irradiated RG2 cells treated with 750 nM of VE822-loaded PEG-poly(PDL-co-DO) or free VE822 (Fig. S3). pChk1 protein levels of irradiated RG2 cells as depicted in Figure 3 were measured by normalizing to Vinculin and total Chk1 levels (Fig. S4).

Animals in the single-fraction groups were irradiated 24 h after CED of the drug, while animals in the 5-fraction RT groups were irradiated starting 48 h after drug delivery. All animals were anesthetized prior to RT using a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg), injected intraperitoneally. Animals were placed in a custom plexiglass holder with molded lead shielding to collimate the beam, allowing for selective cranial irradiation. The animals were irradiated in a XRAD 320 Cabinet X-ray irradiator (Precision Xray, CT, USA). In-depth dosimetry was performed using thermoluminescent dosimeter to ensure that the entire cranium was treated with 10 Gy per fraction for the single-dose groups or 3 Gy per fraction of radiation for the 5-fraction groups. Animals in the 5-fraction groups were treated on consecutive days, spaced 24 h apart for a total of 5 d. The animals’ health status were monitored daily, and decision to euthanize was made after either a 15% loss in body weight or when it was humanely necessary due to clinical symptoms from tumor progression.