Gammacell 3000 elan

Human U-2 OS, HeLa, and HEK293T cells were obtained from ATCC. Cell lines were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1% streptomycin/penicillin antibiotics (Wisent) and 10% fetal bovine serum (Gibco). Cells were grown at 37°C in a 5% CO₂ humidified atmosphere. Cells were regularly tested to ensure the absence of mycoplasma contamination. For treatments, HU (Bioshop), CPT (Alfa Aesar), mitomycin C (MMC) (Tocris Bioscience), cycloheximide (CHX) (Biobasic), MG132 (Calbiochem), VE-821 (ATRi), KU55933 (ATMi), and NU7441 (DNA-PKi) (Selleck Chemicals) were used as indicated in the corresponding figure legends. γ-irradiation was performed in a Gamma cell 3000 Elan (Best theratronics) and UV-C irradiation was done using a luminometer-calibrated Stratalinker 2400 crosslinker (Stratagene).

Female NOD/Rag1^null/IL2Rγ^null (NRG) mice were obtained from The Jackson Laboratory (JAX, Bar Harbor, ME, USA) and bred in-house under pathogen-free conditions. Mice were transplanted with CB CD34⁺ cells as previously described [19 (link)]. Briefly, 5 weeks old mice were irradiated (450 cGy) using a [¹³⁷Cs] column irradiator (Gammacell 3000 Elan; Best Theratronics, Ottawa, ON, Canada) and 4 h later transplanted with 2.0 × 10⁵ human CB-CD34⁺ cells by intravenous (i.v.) injection. To generate humoral responses against HCMV-gB, humanized mice were immunized at 6, 7, 10 and 11 weeks after HCT subcutaneously (s.c., near the anatomical regions of the inguinal and axillary lymph nodes) with 5.0 × 10⁵ iDCgB cells. To stimulate immune responses against of EBV, humanized mice were immunized at 10, 11, 14, 16 and 18 weeks after HCT with 5 × 10⁶ EB-VLPs injected i.v.

Exponentially growing cells (30–40% confluent) were irradiated fully immersed in medium with 6 Gy using Cesium-137 γ-rays (Gammacell 3000 Elan, Best Theratronics, Kanata, ON, Canada). After irradiation, cells were re-incubated in fresh medium.

U2OS, HeLa, HEK293T, TOPORS^+/+ and TOPORS^−/− MEFs were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, USA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen). Cells were maintained in a humidified incubator containing 5% CO₂ at 37°C. All cells were obtained from the American Type Culture Collection (Manassas, VA, USA). TOPORS^+/+ and TOPORS^−/− mouse embryonic fibroblast (MEF) cells were obtained from Dr Eric H. Rubin (36 (link)). To induce DNA damage, exponentially growing cells were treated with ¹³⁷Cs γ-ray at a dose of 2 or 5 Gy (Gammacell 3000 Elan; Best Theratronics, Ottawa, Canada), 2 mM hydroxyurea (HU, Sigma-Aldrich, USA) and then allowed to recover at 37°C for indicated time points.

NRG mice were obtained from The Jackson Laboratory (JAX, Bar Harbor, ME) and bred in-house under pathogen-free conditions. Female mice were used for all experiments (19). Briefly 5 weeks-old mice were sub-lethally irradiated (450 cGy) using a [¹³⁷Cs] column irradiator (Gammacell 3000 Elan; Best Theratronics, Ottawa, Canada). Four hours after irradiation, 2.0x10⁵ human CB-CD34⁺ cells were injected as described before for HSCT [19 (link)]. CB units were tested prior to experiments for humanization potential. Only CB units which resulted into >20% hCD45 (BL) at week 10 post-HCT were used in studies. Cryopreserved and quality-controlled iDCgB were thawed and injected into NRG at weeks 6, 7, 10 and 11 after HCT. Mice were immunized with a total dose of 5.0x10⁵ iDCgB cells injected s.c., near the anatomical regions of the inguinal and axillary lymph nodes. Humanized NRG mice were infected with HCMV as described before (16). Briefly, at week 17 post HCT, mice were treated s.c. with 150 ng hG-CSF/mouse/day (Granocyte, Kohlpharma GmbH, Merzig, Germany) for 3 days, injected i.p. with 1.0x10⁶ MRC-5 cells previously infected with TB40-GLuc (MOI 1) and treated for G-CSF for additional 2 days. For HCMV reactivation, mice were treated s.c. with 2,5 μg hG-CSF/mouse/day for 7 days from weeks 24 and 25 post-HCT.

Mice received a single dose of 4.5Gy to the brain (and ≤1 Gy to the shielded body) on different days after surgery using a Gammacell 3000 Elan irradiator (Best Theratronics), which irradiates the content of a steel cylinder with γ-rays from a sealed Cesium-137 line source as described previously [28 (link)]. Briefly, tumor-bearing mice were anaesthetized with ketamine/xylazine (100/15mg/kg) and placed in an upright position in a 50mL Falcon tube without a tip to facilitate breathing. The tube was placed inside 2cm thick custom-built lead shielding that exposes the head to radiation whilst shielding the body from the ears down (see Fig 1).

Rat intestinal epithelial cell line IEC-6 was obtained from the Korean Cell Line Bank (Seoul, South Korea) and maintained in DMEM with 10% FBS and 1% penicillin–streptomycin. Human recombinant proteins were purchased from Prospec (East Brunswick, NJ, USA). A total of 8–9-week-old male C57BL/6 J mice (Central Lab Animal Inc., Seoul, South Korea) were housed at room temperature under 12-h light–dark cycle and allowed access to water and chow ad libitum. All animal experiments were approved by the Institutional Animal Care and Use Committee at the Korea Advanced Institute of Science and Technology (KA2015-35). Mice and cells were irradiated at a dose rate of 2.16 Gy/min using a Cs-137 irradiator (Gammacell 3000 Elan, Best Theratronics, Ottawa, ON, Canada). Irradiation was performed without anesthesia on mice held in a 50-ml conical tube on a rotating turntable. Dosimetric quality assurance was performed using nanoDots (Al2O3:C) optically stimulated luminescence dosimeters (Landauer, Glenwood, IL, USA), which were read using a MicroStar OSL reader (Landauer, Glenwood, IL, USA).