X rad 320 x ray biological irradiator

TRAMP-C1 murine prostatic adenocarcinoma cells (p53−/−, androgen-independent) were purchased from the ATCC (Bethesda, Maryland, USA). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies, Germany) supplemented with 10 % (v/v) fetal calf serum (FCS; Biochrom, Berlin, Germany) and maintained in a humidified incubator at 37 °C and 5 % CO₂ (C200, Labotect Incubator, Goettingen, Germany). Cells were irradiated using an X-RAD 320 X-Ray Biological Irradiator with a MIR-324 X-ray tube (Precision X-Ray Inc., North Branford, USA). Cell number and viability was quantified by counting cells using CASY cell counter (Innovatis, Reutlingen, Germany).

X-ray irradiation was performed at room temperature with X-RAD 320 X-Ray Biological Irradiator (Precision X-ray, North Branford, CT, USA) using 320 kV, 12.5 mA, a 1.65 mm aluminum filter and a dose rate of 3.40 Gy/min with the cells in a distance of 50 cm were or the X-ray generator ISO-DEBYEFLEXVOLT 3003 with an industrial X-ray tube 160 M1/10-55 with installed shutter (15b) operated at 35kV and 80mA (GE Sensing & Inspection Technologies GmbH & Co.KG, Ahrensburg, Germany). Control cells were kept at room temperature for the same period of time as their irradiated counterparts.
Cells were treated with AAPA (40 µM, Merck KGaA, Darmstadt, Germany), BSO (200 µM, Sigma-Aldrich, St. Louis, MO, USA), MK2206 (4 µM, Selleckchem, Houston, TX, USA) or 2DG (10 mM, Sigma-Aldrich, St. Louis, MO, USA) 1 h prior to IR. Rescue experiments (as described in (63 (link))) were performed using 4 mM glutathione ethylester (GEE; Cayman Chemical, Ann Arbor, MI, USA) which was applied 1 h prior to IR. For long term colony formation assays, the drug concentration was reduced for AAPA to 4 µM and BSO 20 µM.

In the hormesis study, cells were irradiated with 20, 50, 75, 100, 200, 1000, and 3000 mGy X-rays at room temperature. In the adaptive response study, cells were exposed to an optimum adaptive dose (D1) that was confirmed in the hormesis study, with or without a challenge dose (D2) of 5 Gy. The interval between D1 and D2 was determined based on the results of the WST-1 assay and cell cycle analysis. Control groups were treated in a similar manner but without irradiation. After an additional 24 h of incubation, cells in each group were collected for analysis. Cells were irradiated using an X-RAD 320 X-Ray Biological Irradiator (Precision X-Ray, North Branford, CT, USA). Dose rates of 12.5 mGy/min and 500 mGy/min were used for LDR and HDR, respectively.