Cells were seeded at densities ranging from 300 to 10,000 in 6 cm dishes and incubated for 12 hr to allow attachment. Cells were then treated with NCS (stock concentration 37 μM diluted to 2 μM in 20 mM sodium citrate buffer, pH 4.0) at concentrations ranging from 0.25 nM to 2 nM for HCT116 cells and 2 nM to 6 nM for HeLa cells for 6 hr. Following treatment, cells were incubated in fresh medium for 8-9 days to form colonies. Colonies were fixed with 100% methanol for 10 min, stained with 0.5% crystal violet in 20% methanol for 10 min, washed under tap water, air dried and counted manually. Plating efficiency (PE) was calculated as the number of colonies formed/number of cells seeded *100 for each dose. Surviving fraction (SF) was calculated as PE of treated/PE of control *100. Dose-modifying factor (DMF) was calculated as IC90 of control/IC90 of the mutant cell line. For experiments using ionizing radiation, cells were irradiated using a MDS Nordion Gammacell 40 research irradiator (ON, Canada), with a 137Cs source. For experiments with KU-60019, NU-7441, olaparib (AZD-2287) and veliparib (ABT-888), the respective inhibitor was added 1 hr prior to NCS treatment and left in the medium during and 24 hr after NCS treatment. Inhibitors were from Selleck Chemicals (Houston, TX).
Gammacell 40 research irradiator
Manufactured by Nordion
Sourced in Canada
The Gammacell 40 is a research irradiator designed for the controlled irradiation of samples. It utilizes a radioactive source to provide a uniform radiation field for the exposure of various materials or biological specimens. The Gammacell 40 is intended for use in research and scientific applications where controlled radiation exposure is required.
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3 protocols using gammacell 40 research irradiator
1
Clonogenic Survival Assay for NCS
2
Immunofluorescence Assay for γ-H2AX
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Human lung adenocarcinoma epithelial cells A549 and H-1299 were obtained from American Type Culture Collection and grown as recommended. All cell lines were used within 6 months after resuscitation. For γ-H2AX immunofluorescence, H-1299 or A-549 cells were seeded on coverslips in 6-well dishes at a density of 1 × 105 cells/well and allowed to attach for 24 h. These were then treated with the ABT-888, DETA or combined for 4 h and then exposed to a single dose of IR (MDS Nordion Gammacell 40 research irradiator, ON, Canada). Then cells were incubated for 4 h and 24 h post-irradiation. After washing in PBS three times, cells were fixed in 4% formaldehyde for 20 min and permeabilized with 0.2% TritonX-100 in 5% BSA/PBS for 30 min. The coverslips were then incubated in primary antibodies placed on parafilm strips and incubated at RT for 1 h. The coverslips were washed in PBS three times for 5 min each. They were then incubated with secondary antibodies at RT for 1 h. The coverslips were washed with PBS three times for 5 min each, air-dried and then mounted in Fluoroshield mounting medium with DAPI (Abcam) and viewed using a fluorescence microscope.
3
Localized Mouse Irradiation Techniques
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Two systems were used for the irradiation of mice. A Cesium-137 (137Cs) source was used for whole body mouse irradiation (Gammacell 40 research irradiator, MDS Nordion, Toronto, ON, Canada), with mice receiving a single whole body dose of 10 Gy. The small animal radiation research platform (SARRP, Xstrahl Inc., Suwanee, GA, USA) was used for localized stereotactic mouse irradiation to 10 Gy in a single fraction. SARRP irradiation of the mouse colon was performed on anesthetized mice (2% isoflurane in oxygen) using 220 kV photons at 13 mA targeted to the midline mouse colon. Onboard imaging using cone beam CT (65 kV, 1 mA) allowed for tissue density calculation for dosimetry and anatomic delineation of tissues. Beams were targeted from the mouse rectum to 3 cm cranial using 2 orthogonal beam positions at each of 3 isocenters 1 cm apart using a 1 cm square collimator. The first isocenter was selected to cover the entire rectum, and each subsequent isocenter was placed exactly 1 cm cranially. Two orthogonal beams were used at each beam location at 45° and −45° to prevent high surface dose deposition and avoid attenuation of the beam by the spine. SARRP irradiation of xenograft tumor-bearing mice with tumors on their rear flanks was performed on anesthetized mice using a single beam position at 0° and a 1 cm square collimator.
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