For growth curve analysis, cells expressing H2B-GFP were treated with 0, 2, 6 or 8 Gy of radiation in a Gammacell 3000 (Best Theratronics) and seeded in a 96-well plate. Cell proliferation was followed using the IncuCyte ZOOM or IncuCyte S3 Live-cell Imaging System (Sartorius, Göttingen, Germany) using a 10× objective. The number of cells was evaluated using Incucyte® Software by the number of green fluorescent nucleus of cells expressing H2B-GFP in at least two wells per condition. Experiments were performed at least three times.
Gammacell 3000
Manufactured by Best Theratronics
Sourced in Canada
The Gammacell 3000 is a self-shielded gamma irradiator designed for research and medical applications. It utilizes a Cobalt-60 source to generate gamma radiation. The Gammacell 3000 provides a uniform dose distribution within the irradiation chamber, which can accommodate a variety of sample sizes.
Automatically generated - may contain errors
Lab products found in correlation
Incucyte zoom, by Sartorius (1 mentions)
Incucyte s3 live cell imaging system, by Sartorius (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Roswell park memorial institute (rpmi), by Merck Group (1 mentions)
Fetal calf serum (fcs), by Biosera (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
6 protocols using gammacell 3000
1
Growth Curve Analysis of Radiation-Treated Cells
2
T Cell Proliferation Assay with Staphylococcal Antigens
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Anonymized buffy coats were obtained from healthy blood donors at the Irish Blood Transfusion Service, Dublin, Ireland. Peripheral blood mononuclear cells (PBMCs) were isolated, and CD4+ cells were purified (CD4+ T cell isolation kit; Miltenyi Biotec) and labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) as previously described (5 (link)). PBMCs were gamma irradiated at 30 Gy with a 137Cs source (Gammacell 3000 Best Theratronics). CFSE-labeled CD4+ cells (1 × 105) were cocultured with irradiated PBMCs (APCs) (1 × 105) with cRPMI alone (negative control), staphylococcal enterotoxin A (100 ng/ml [positive control]), heat-inactivated S. aureus (1 μg/ml ≈ 1 × 107 CFU/ml), or purified staphylococcal antigens (0.88 μM). cRPMI comprised RPMI (Sigma), 10% (vol/vol) fetal calf serum (Biosera), 100 mM l -glutamine (Gibco), and 100 μg/ml penicillin-streptomycin (Gibco). T cell proliferation and cytokine production were assessed on day 10 by flow cytometry.
3
Clonogenic Assay for Irradiated OSCC Cells
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OSCC cells were seeded at a density of 200,000 cells per dish in 60-mm dishes and cultured overnight before pretreatment with 10 µM LPA for one hour prior to irradiation. In experiments using inhibitors, cells were pretreated with 10 µM LPA for one hour prior to the addition of 10 µM NS-398 for a further one hour before irradiation. Media were changed prior to irradiating the cells with 0-8 gray (Gy) gamma using the Gammacell 3000 (Best Theratronics, Ottawa, Canada). Irradiated cells were harvested immediately by trypsinisation. Cells were counted and 300-500 cells were plated in triplicate for clonogenic survival assays. The cells were incubated for 12 days with media changed every four days and colonies stained with 0.1% (w/v) crystal violet. Colonies with >50 cells were counted as one colony. The plating efficiency (PE) and survival fraction (SF) were calculated using the following equations:
4
Ionizing Radiation and Targeted Therapies
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Ionizing radiation treatment was performed with the Gammacell 3000 (Best Theratronics, Ottawa, ON, Canada) at defined doses (0, 2, 4, 6, 8 or 10 Gy). We purchased ABT-199 (Venetoclax) that inhibited BCL2 or ABT-263 (Navitoclax) for BCL2/BCLxl/BclW from Medchem and APExBIO. Both drugs were prepared with Dimethyl sulfoxide (DMSO) and DMSO from Sigma was used for vehicle alone in control condition. In combination treatment, pre-irradiation conditions represented cells that were exposed to 8 Gy of radiation five days before adding drugs while control cells were seeded two to three days before drug addition.
5
Gamma Irradiation of 3D Spheroids
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Two days after seeding, fresh media were added to each channel before irradiation of spheroids directly on chip using the Gammacell 3000 irradiator (Best Theratronics, Ottawa, ON, Canada) at defined doses (0, 2, 4 and 8 Gy).
6
Leukoreduced and Irradiated RBC Transfusion
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RBCs were collected in bags containing citrate-phosphate-dextrose-adenine-1. Prior to transfusion, RBCs underwent post-storage leukoreduction using an RCM1 filter (Haemonetics, Braintree, MA) until March 2012, and thereafter RBCs underwent pre-storage leukoreduction using an RC2VAE filter (Haemonetics). Both filters reduce the number of residual leukocytes per RBC unit to <210 5 . All RBCs were further gamma-irradiated using our on-site cesium-137 irradiators: an IBL 437 (CIS, Bedford, MA) was used until February 2008, and thereafter, a GammaCell 3000 was used (Best Theratronics, Ottawa, Ontario, Canada). The irradiation duration was modified based on dose distribution mapping performed by outside experts so that the absorbed-dose at the center of the target was 25 Gy, with a minimum of >15 Gy and maximum of <50 Gy throughout the target (Supplementary Fig. S1). The use of RBCs stored for >24 hours after irradiation was prohibited because of concerns of massive RBC storage lesion (e.g. hyperkalemia) without the consideration of plasma washing. 23 Compatibility was routinely evaluated using ABO/Rh typing, testing recipient serum for clinically important alloantibodies, and serologically cross-matching donor RBCs with recipient serum. Our blood bank delivered RBCs to the operating room using the first-in-first-out principle to consume older RBCs before newer one. 6
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