Irradiation was conducted using an Xstrahl RS225 cabinet with a single dose of 2, 4, 6 or 8 Gy (Gy). X-rays were delivered at 195 kV, 10 mA at a dose rate of 0.87 Gy/min. The cabinet was fitted with a 0.5-mm copper filter and used at a 48.4-cm focus-to-skin distance. Sham-irradiated cells were used as controls. Cells/spheroids were treated with 1 µM of IQ10 for 4 h under normoxia prior to irradiation. For 2D hypoxic experiments, cells were treated with clonogenic IC50 doses of IQ10 for 48 h under 1% O2 and then irradiated. Cells/spheroids were then trypsinised and plated for clonogenic survival assay as described above. Survival curves were fitted to a linear-quadratic model using the software CS-CAL (German Cancer Research Centre). The mean values of parameters α, β, α/β and SF2 (surviving fraction at 2 Gy) were calculated from the fitted curves. The SER was calculated as the radiation dose needed for radiation alone divided by the dose needed for IQ10 plus radiation at a SF of 1%.
Rs225
Manufactured by Xstrahl
Sourced in United Kingdom
The RS225 is a versatile x-ray irradiator designed for laboratory research applications. It delivers controlled doses of x-ray radiation to experimental samples or cell cultures. The system features a robust, shielded enclosure and a precision dose delivery mechanism to ensure accurate and reproducible irradiation results.
Automatically generated - may contain errors
12 protocols using rs225
1
Radiosensitization by IQ10 under Normoxia and Hypoxia
2
Clonogenic Survival of Cancer Cells
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Cells were seeded in 25 cm2 flasks at 105 cells/flask. When cultures were in exponential growth phase, medium was removed and replaced with fresh medium (including 1% or 10% serum) containing drug. For radiation treatment, cells were irradiated using an X-Strahl RS225 x-ray irradiator (Xstrahl Limited, Surrey, United Kingdom) at a dose rate of 1.6 Gy per minute. Cells were then incubated for 24 h at 37ºC in 5% CO2. After treatment, cells were counted and seeded for clonogenic survival assay as previously described.15 (link) Cells were incubated at 37°C in 5% CO2 for up to 13 days. LNCaP cells did not form clonogens under similar conditions and so were not used for clonogenic assay. Colonies were fixed in methanol, stained with crystal violet solution, and colonies of at least 50 cells were counted.
3
Ionizing Radiation and Immune Checkpoint Inhibitors Impact on OAC Cells
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A cell counting kit-8 (CCK-8) viability assay was used to determine the impact of ionising radiation on the viability of OE33P and passage matched OE33R cells. The impact of anti-PD-1, and anti PD-L1 therapies in isolation, and dual ICB with and without radiation, at both hypofractionation and bolus dosing of clinically relevant doses on the viability of OE33P and R cells was also assessed using a CCK-8 assay. OAC cells (5 × 103) were adhered in a 96 well plate at 37 °C, 5% CO2 overnight. Cells were treated with bolus dosing or three consecutive fractionated doses of radiation with an interval of 24 h using the X-Strahl RS225 irradiator. In addition to this, the cancer cells were treated with and without radiation in the absence or presence of pembrolizumab (10 μg/mL), atezolizumab (10 μg/mL), nivolumab (10 μg/mL) or combination atezolizumab (10 μg/mL) and nivolumab (10 μg/mL), or dual atezolizumab (10 μg/mL) and pembrolizumab (10 μg/mL). All of the data were analysed from three independent experiments (Supplementary material ).
4
Irradiating Cell Cultures with X-rays
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Cultures were irradiated in tissue culture flasks at room temperature with 195 kV X-rays at a dose rate of 1.6 Gy/min in an Xstrahl RS225 cabinet.
5
Whole-Body X-Ray Irradiation in Mice
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The mice were whole-body irradiated with doses of 0.5 Gy, 1 Gy or 3 Gy using the X-ray research irradiator cabinet RS225 or the irradiation platform SARRP (X-Strahl, Ratingen, Germany). The sham-irradiated mice were transported to the irradiation systems in the same way as the irradiated mice and placed next to the irradiation field to ensure that they were exposed to a similar stress level.
6
Low-LET X-ray Irradiation of Cells
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Cells were exposed to low LET (0.3-3.0 keV/μm) X-rays using the Gulmay X-ray source RS225 (X-Strahl, Surrey, UK) at DLR Cologne. The X-ray tube was adjusted to 200 kV and 15 mA. To eliminate soft X-rays, a Copper (Cu) filter with a thickness of 0.5 mm was used. Dose and dose rate were determined using the dosimeter UNIDOS webline with the ionization chamber TM30013 (PTW, Freiburg, Germany). The distance of the sample from the X-ray source was set to 450 mm to provide a constant dose rate of 1.0 Gy/min. The temperature inside the X-ray chamber was kept at 37 • C and samples were transferred after exposure to the incubator. As the X-ray source was located above the samples, cells in strips were exposed in horizontal position below the exit window.
7
Orthovoltage X-Ray Irradiation of Tumor-Bearing Mice
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Radiation was delivered to tumor-bearing mice using a cabinet orthovoltage X-ray biological irradiator, X-RAD 320 (Precision X-Ray, Inc.). Local radiation to the tumor site was delivered after immobilization and shielding of mice using custom lead jigs that exposed only the tumor + ~5 mm margin. Radiation for in vitro experiments was delivered using an RS225 (Xstrahl) cabinet orthovoltage irradiator and was performed at least 24 h after plating the cells. Media was replaced immediately after radiation delivery.
8
Radiation-induced DNA Damage Inhibition
9
Multimodal Radiation and Erlotinib Therapy
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Experiments were performed using the A549 human bronchial carcinoma cell line (ATCC CCL-185, Manassas, Virginia, USA), the FaDu head and neck tumor cell line (ATCC, HTB43), and HSF7 normal fibroblasts [23 (link)]. Irradiations were performed using the X-ray cabinet RS 225 (X-Strahl, Surrey, United Kingdom). The voltage was set to 200 kV, with a current of 15 mA (dose rate = 1 Gy/min at a 49 cm distance from the X-ray tube). The X-ray beam was hardened with a 0.5 mm removable copper filter. Dosimetry was performed with a farmer chamber (PTW, Freiburg, Germany). Irradiation was conducted at 37°C. Erlotinib was purchased from Selleck (Houston, Texas, USA) and EGF was purchased from Sigma-Aldrich (St. Louis, Missouri, USA).
10
Cytotoxicity and Radiosensitization Assay
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The NET cells were seeded and treated for 24 h with 5-fluorouracil, decitabine, or tacedinaline at inhibitory concentration (IC) 10 or IC20 for each drug before being irradiated at doses of 0-6 Gy using a XStrahl RS225 radiation cabinet (XStrahl Inc.) and further cultured for 14 d (BON1 cells) or 21 d (QGP1 cells). After that, cells were fixed with 70% ethanol and stained with 0.3% methylene blue. Colonies with more than 50 cells were counted, and the surviving fractions (SFs) were calculated versus the plating efficiency. The survival curves were established by the linear-quadratic model, and the sensitization enhancement ratio (SER) was measured as follows: SF 5 1 2 (1 2 exp[2D/D 0 ]) N ; and SER 5 SER SF2 5 control group SF2 value/treatment group SF2 value.
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