The colony-forming assay was performed to determine radiosensitivity. Cells were plated in 6-well plates and allowed to adhere overnight before exposure to radiation at the indicated doses and a dose rate of 5 Gy/min using 6-MV X-rays generated by a linear accelerator (Varian 2300EX, Varian, Palo Alto, CA). After incubation for 10–14 days, the cells were stained with 0.5% crystal violet in methanol. Then the colonies (clusters of > 50 cells) were counted under a microscope. Survival data from different experiments were pooled, after which survival curves were fitted and analyzed by using the linear-quadratic model (LQ). The sensitizing enhancement ratio (SER) and oxygen enhancement ration (OER) were calculated as described previously [22 (link), 33 (link)].
Varian 2300ex
Manufactured by Agilent Technologies
Sourced in United States
The Varian 2300EX is a high-performance gas chromatograph designed for laboratory use. It features a rugged construction and advanced electronics for reliable operation. The 2300EX is capable of separating and analyzing a wide range of chemical compounds.
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16 protocols using varian 2300ex
1
Radiosensitivity Determination via Colony-Forming Assay
2
Dose-dependent X-ray Irradiation of Transfected Cells
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Transfected cells were exposed to various doses of 6-MV X-ray (0 Gy, 2 Gy, 4 Gy, 6 Gy and 8 Gy) using a linear accelerator (Varian 2300EX, Varian, Palo Alto, CA, USA) at a dose rate of 200 cGy/min.
3
Candesartan Enhances Tumor Radiosensitivity
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All experimental procedures were approved and overseen by Southern Medical University Institutional Animal Care and Use Committee, and were performed in accordance with the guidelines and regulations for animal experiments set down by Southern Medical University. Four-week-old female BALB/C nude mice were used for all animal experiments. CNE2, 5–8F or MDA-MB-231 tumor cells (5 × 106 cells) were inoculated s.c. into the right upper leg of each mouse. Tumors were allowed to grow for 8 days before randomization and treatment. To explore the influence of candesartan in radiosensitivity of tumors, the right upper leg tumor site was irradiated with 10 Gy on day 8 and candesartan (5 mg/kg, i.p., Takeda Pharmaceutical Company Limited, Japan) was given daily for 3 days before radiation and 3 days after radiation. Irradiation was performed at room temperature using 6-MV X-rays generated by a linear accelerator (Varian 2300EX; Varian, Palo Alto, CA). Tumors were measured every three days from the start of irradiation for 3 to 8 weeks, as indicated in the results. Tumor volume was calculated as length × width2 × 0.5.
4
Clonogenic Cell Survival Assay
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After irradiation (dose rate: 5 Gy/min) using a 6-MV X-ray generated by a linear accelerator (Varian 2300EX, Varian, Palo Alto, CA) at the indicated dose, the plates were further incubated for 10–14 days, then the cells were fixed with 10% methanol and stained with 1% crystal violet in 70% ethanol. Colonies (>50 cells) were counted under a light microscope. The curve of survival fraction was derived from the multi-target single-hit model: SF=1-(1-exp(-x/D0))^N.
5
Clonogenic Assay for Radiosensitivity
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For clonogenic survival assays, CNE‐1 and CNE‐2 cells were seeded into six‐well plates at specific cell densities in triplicate, after 48 h of transfection of miR‐24 mimic or SP1 siRNA, followed by exposure to the indicated doses of radiation (0, 2, 4, 6, 8, or 10 Gy) by using 6 MV X‐rays generated by linear accelerators (Varian 2300EX; Varian, Palo Alto, CA) at a dose rate of 3 Gy/min. Following 10–16 days of incubation at 37°C, the cells were fixed using 100% methanol and stained using 1% crystal violet (Sigma ‐ Aldrich Co., St. Louis, MO, USA). Microscopic inspection (Olympus IX71; Olympus, Tokyo, Japan) was used for the colonies containing ≥50 normal‐looking cells. As described previously 20 , the surviving fraction was calculated. The multitarget single‐hit model was fitted to the data to generate survival curves using the formula: SF = 1‐(1‐e‐D/D0)N. The sensitization enhancement ratio with a survival fraction of 10% (SER10) was calculated later. Each experiment was independently performed at least three times.
6
Radiosensitization Effect of miR-101 and STMN1 siRNA
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CNE-2 and 5-8F cells were pretreated by either miR-101 mimic or STMN1 siRNA transfection for 48 h and subsequently seeded onto six-well plates in triplicate at specific cell densities, followed by exposure to the indicated doses of radiation (0, 2, 4, 6 or 8 Gy) using 6 MV X-rays generated from linear accelerators (Varian 2300EX; Varian, Palo Alto, CA, USA) at a dose rate of 3 Gy/min. Following 10–14 days of incubation at 37°C, the cells were fixed using 100% methanol and stained using 1% crystal violet (Sigma-Aldrich). Colonies containing ≥50 normal-appearing cells were counted via microscopic inspection (Olympus IX71; Olympus, Tokyo, Japan). The surviving fraction was calculated as described previously (20 (link)). The multi-target single-hit model was fitted to the data to generate survival curves using the formula: SF=1−(1−e−D/D0)N. The sensitization enhancement ratio at a survival fraction of 10% (SER10) was subsequently calculated. Each experiment was independently performed ≥three times.
7
Clonogenic Assay for Radiation Sensitivity
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Equal numbers of cells were seeded in six-well culture plates (pretreated with siRNAs or oligonucleotides, or plasmid transfected for 48 h) in triplicate and exposed to the indicated doses of irradiation using 6-MV X-rays from linear accelerators (Varian2300EX; Varian, Palo Alto, CA, USA) at a dose rate of 5 Gy/min. After incubation at 37 °C for 14–21 days, the plates were fixed with 100% methanol and then stained with 1% crystal violet. Colonies containing >50 cells were counted by microscopic inspection. The surviving fraction (SF) was calculated. A multitarget single-hit model was fitted to the data to generate survival curves using the following formula: SF = 1 − (1 − e−D/D0)N.
8
Establishment of Radiation-Resistant Hepatocellular Carcinoma
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Human HCC cell line HepG2 (BNCC338070) was provided by BeNa Culture Collection (BNCC, China). HepG2 cells were cultured in DMEM +10% fetal bovine serum (FBS) medium and placed in an incubator at 37°C with 5% CO2. The establishment of radiation-resistant HCC cells was performed as per the method described by Chen et al. [19 (link)]. Specifically, the HepG2 cells were treated with 6-MV X-rays generated by a linear accelerator (Varian 2300EX, dose rate of 2 Gy/min; Varian, USA). The cells were cultured after the initial radiation of 2 Gy and subcultured twice. The surviving cells were then exposed to a series of gradually increasing radiation doses (4, 6, 8, and 10 Gy) twice. The total radiation dose was 50 Gy, and the whole selection process was finished within 6 months. The final surviving cells were defined as HepG2/RR (radiation-resistant). We used parental HepG2 cells as controls and were defined as HepG2/RS (radiation-sensitive). HepG2/RR and HepG2/RS were cultured at 37°C for 24 h before further analyses.
9
Clonogenic Assay for Radiation Sensitivity
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After 48 h of transfection, cells were transferred to complete medium and seeded on 6-well plates in triplicate and exposed to radiation at the doses indicated using a 6-MV X-ray generated by a linear accelerator (Varian 2300EX at a dose rate of 5 Gy/min; Varian MedicalSystems, Palo Alto, CA). After incubation at 37 °C for 14 days, cells were fixed in 100% methanol and stained with 1% crystal violet. Colonies containing >50 cells were counted under a light microscope (Olympus, Japan). The surviving fraction was calculated as previously described12 (link). Each assay was repeated in three independent experiments.
10
Colony Formation Assay of IR Survival
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Equal quantities of cells were seeded in plates in triplicate. The cells were then exposed to IR at the indicated doses (Varian2300EX, Varian, Palo Alto, CA). After incubation for 10–14 days, the cells were fixed and stained with 4% paraformaldehyde and 1% crystal violet, respectively. Colonies with more than 50 cells were counted using microscopy. Survival curves were generated using the multitarget single-hit model.
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