Irradiation. Irradiation of components containing lymphocytes is performed in order to render those cells incapable of reproduction and to reduce the likelihood of transfusion associated graft vs. host disease (TA-GVHD). TA-GVHD is a rare but devastating complication of transfusion that has a very high mortality rate and no routinely available effective treatment. Prevention is the best management of TA-GVHD. AABB Standards require irradiated blood be provided to recipients at risk of TA-GVHD, recipients receiving blood products from relatives, and recipients receiving HLA- matched or crossmatched blood components. a. Irradiated cellular blood products should be given to, but not limited, to patients with the following: • Transfusions from blood relatives • HLA-matched transfusions • Granulocyte transfusions • Intrauterine transfusions • Congenital immunodeficiency (ie DiGeorge Syndrome) • BMT and stem cell transplantation • Hodgkin disease • NHL • Therapy with purine analogue drugs
Irradiation. High-energy HZE particle irradiations were performed in Brookhaven, NY at the NASA Space Radiation Laboratory (NSRL). The X- ray (low-LET) exposures were conducted at Emory University using an X-RAD 320 biological irradiator (Precision X-Ray, North Branford, CT). Three biological replicate cultures containing either 1x10e6 cells (for acute time point) or 2x10e5 cells (for continuous culture) in T-25 flasks were irradiated independently with 0, 0.1, 0.3 or 1.0 Gy 56Fe ions (Beam energy: 600 MeV/u; dose rate for the 0.1 Gy dose was 0.1Gy/min, for the 0.3 Gy dose, 0.3 Gy/min, and for the 1.0 Gy dose, 1Gy/min.) or with 0.0, 0.3, 1.0 Gy 28Si ions (Beam energy: 300 MeV/u; dose rate for the 0.3 Gy dose was
Irradiation. Exponentially growing cells were seeded in 10-cm dishes or 6-well plates. Twenty-four hours later, the cells were exposed to recombinant viruses AdMLP.apoptin or AdCMV.LacZ at an MOI of 5 at 37ºC. Fresh medium (2 mL) was then added to each well and incubation was continued for 24 hrs. One day after the viral infection, cells were irradiated using a 6 MV linear accelerator. Doses were given in a single fraction of 2–6 Gy. After irradiation, the dishes were immediately returned to the incubator. Cytochrome c assay. First treated cells were subcellularly fractionated. The cell monolayer (106 cells/10-cm dish) was rinsed twice with ice-cold PBS, scraped and centrifuged at 2000 g for 5 min. The cells were resuspended in cell extraction buffer (300 mM sucrose, 10 mM Hepes [pH 7.4], 50 mM KCl, 5 mM EGTA, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 100 µM cytochalasin B), left on ice for 30 min, and homogenized by means of vortexing the lysates. Unbroken cells and nuclei were pelleted by centrifugation for 5 min at 2000 g. Heavy membranes were removed from the resulting supernatant by centrifugation for 5 min at 14000 g. The resulting supernatant is the crude cytosolic fraction. All samples were frozen in liquid nitrogen and stored at -80° C until analysis by sodiumdodecylsulfide-polyacrylamide gel electrophoresis (SDS-PAGE). Next, the cytoplasmic cytochrome c levels were determined. Equal amounts of cytosolic protein extract were boiled in reducing sample buffer for 5 min, loaded in a lane of a sodiumdodecylsulfide-15% polyacrylamide (SDS-PAA) gel, and electroblotted onto Immobilon-P membranes (Millipore, Bedford, MA, USA). The membranes were blocked for 1 hour in PBST-buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20) containing 5% nonfat dried milk. Subsequently, the blots were then incubated during overnight with monoclonal antibody 7H8.2C12 (Pharmingen; dilution 1:1000) to detect cytochrome c. Following overnight incubation, membranes were washed and incubated for 1 h at room temperature with secondary antibody. Membranes were washed three more times and incubated for 5 min at room temperature with Super Signal HRP substrate (Xxxxxx). Positive signals were visualized by enhanced chemiluminescence according to the manufacturer’s protocol (Amersham, Roosendaal, the Netherlands). Caspase-3 analysis. Cells (8 × 105/dish) were grown in 60-mm plates and treated as follows: Cells were collected by gentle scraping, washed once w...
