The goal of these experiments was to determine if BM cells isolated from mice with MCT‐PH were capable of inducing pulmonary hypertensive changes after transplantation into healthy mice (Supporting Information Fig. 1 , Study Protocol). In these experiments, mice received weekly subcutaneous injections of MCT (600 mg/kg, Sigma) or PBS (vehicle) for 4 weeks. One week after the last series of injections, mice were sacrificed for BM cell harvest or for analysis as described below. Whole bone marrow (WBM) cells were isolated by flushing tibiae, femurs, and iliac crests with PBS supplemented with 5% fetal bovine serum (FBS). Cells were strained through a 40 µm cell strainer then centrifuged (300g, 10 minutes) and resuspended in PBS/5% FBS. Cells were counted and viability was determined using trypan blue staining. 2 × 106 WBM cells isolated individually from each mouse were then infused via tail vein into lethally‐irradiated mice (950 centigray, Gammacell 40 Exactor Irradiator, MDS Nordion) so each individual transplant recipient received WBM from only one WBM donor mouse. Mice were sacrificed 4 weeks later for analysis.
Gammacell 40 exactor irradiator
Manufactured by Nordion
Sourced in Canada
The Gammacell 40 Exactor irradiator is a compact, self-shielded gamma irradiator designed for research and development applications. It utilizes a Cobalt-60 radioactive source to provide a uniform radiation field within the irradiation chamber. The irradiator is equipped with a load/unload mechanism and a timer to control the duration of the irradiation process.
Automatically generated - may contain errors
Lab products found in correlation
Cobalt chloride cocl2, by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Dulbecco s modified eagle medium, by Lonza (1 mentions)
U87 atcc htb 14, by American Type Culture Collection (1 mentions)
Bafilomycin, by Merck Group (1 mentions)
Metformin, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (1 mentions)
Fetal calf serum (fcs), by Lonza (1 mentions)
8 protocols using gammacell 40 exactor irradiator
1
Bone Marrow Transplant in Pulmonary Hypertension
2
Tracking Adoptive Immune Cell Therapy
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Recipient CD45.2+ C57BL/6 mice were lethally irradiated (8 Gy) using a 137Cs irradiator (Gammacell 40 Exactor irradiator; MDS Nordion International, Ottawa, ON, Canada). Five hours later, the irradiated mice were injected intravenously with a single‐cell suspension of BMCs (1 × 107) isolated from donor CD45.2+ C57BL/6 mice. The mice were injected intravenously with 3 × 106 iLs harvested from co‐cultures of HSC‐eBMCs +iTECs + IL‐2 + IL‐7 on day 7. Spleen, peripheral blood, small intestine lymph nodes, thymus and bone marrow were harvested from the mice 14 days later, and CD45.1+ iLs were identified and analysed by flow cytometry. For histological analysis, liver, lung, kidney and small intestine were harvested on day 31 after iL injection.
3
Combination Treatment of ABT-737 and Radiation
4
Clonogenic Assay for Glioblastoma Radiation and Temozolomide Sensitivity
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LN18 glioblastoma cells were transfected with siRNA (siCtr or siF), after twenty-four hours, cells were harvested and plated in 6-well plates at different concentration (500, 750, 1000 cells/well for siCtr and 1500, 2500, 5000 for siF) in duplicate. Twenty-four hours later cells were irradiated with an ionizing radiation scale (from 0 to 4 Gy) using the Gammacell 40 Exactor irradiator (Nordion, Ottawa, Canada) or with TMZ dose scale (from 100 to 500 µM). Cells were then incubated for approximately 10 days until colonies were visible with the naked eye without any joining between colonies. Then, plates were washed and cells were fixed with 10% formalin for 10 min, the formalin was removed and cells were covered with 10% crystal violet oxalate (RAL Diagnostics, Martillac, France) for 10 min, plates were rinsed with water until no additional color comes off the plate. Colonies were then counted to calculate the plating efficiency. Plating efficiency (%) = (number of colonies formed/number of cells plated) × 100.
5
Whole Body Gamma Irradiation in Mice
6
Human Glioblastoma Cell Line Cultivation
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Human glioblastoma cell lines U87 (ATCC HTB-14, obtained from the American Type Culture Collection, Manassas, VA, USA), U251, LN18 and SF767 (obtained from C. Simon’s laboratory, UPENN, Philadelphia, PA, USA) were used and routinely maintained in Dulbecco’s Modified Eagle Medium (Lonza, Portsmouth, NH, USA) supplemented with 10% fetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in 5% CO2-humidified incubators as previously described. Cells were tested for mycoplasma contamination by PCR. When indicated, cells were treated with cobalt chloride (CoCl2; Sigma, St Louis, MO, USA) at 100 μM (2 × 50 μM at 24 h and 48 h after transfection) or with MG132 (5 μM for 2 h; Sigma, St Louis, MO, USA) or irradiated at 5 Gy using the Gammacell 40 Exactor irradiator (Nordion, Ottawa, ON, Canada).
7
Glioblastoma Cells Radiosensitization Assay
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U251 and LN18 glioblastoma cells were transfected with siRNA (siScr or si3e); after 48 h, cells were harvested and plated in six-well plates at different concentrations (10, 15 or 20 cells/cm2 for siScr and 20, 30 or 40 cells/cm2 for si3e) in duplicate. 48 h later, cells were irradiated with an IR scale (from 0 to 4 Gy) using the Gammacell 40 Exactor irradiator (Nordion, Ottawa, ON, Canada). Cells were then incubated for ∼10 days until colonies were visible with the naked eye without any joining between colonies. Then, plates were washed and cells were fixed with 10% formalin for 10 min, the formalin was removed and cells were covered with 10% crystal violet oxalate (RAL Diagnostics, Martillac, France) for 10 min; plates were rinsed with water until no additional color comes off the plate. Colonies were then counted to calculate the dose enhancement factor (DEF). DEF is measured as follows: for the same biological effect, the dose on the curve radiation alone (here Scr) is divided by the dose on the curve radiation + treatment (here 3e). A DEF >1 means that the treatment is functioning as a radiosensitizer.
8
Glioblastoma Cell Lines Treated with Compounds
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Four human glioblastoma cell lines, U87 (ATCC HTB-14), LN18 (ATCC CRL-2610), U251 and SF767 (obtained from M. Celeste Simon’s laboratory, University of Pennsylvania, Philadelphia, PA, USA), were used and routinely maintained in Dulbecco’s Modified Eagle Medium (DMEM, Lonza) supplemented with 10% fetal calf serum (Lonza) at 37C° in 5% CO2-humidified incubators and were subcultured once or twice a week as described previously [20 (link)]. Cells were treated or not with the following compounds, 1-10mM metformin (Sigma-Aldrich), 5–100μM temozolomide (Schering-Plough Europe), 10μM bafilomycin (Sigma-Aldrich) and irradiated or not at 5Gy using the Gammacell 40 Exactor irradiator (Nordion).
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