Gammacell 40 irradiator

Bone marrow transplantation was performed as described previous.^{60 (link)} In brief, WT mice at 6 weeks of age underwent whole-body gamma irradiation with 137Cs using a Gammacell 40 irradiator (MDS Nordion, Ontario, Canada). A lead shield was used to protect the head and eyes from radiation. A dose of 8.5 Gy (850 rads) was used to ablate the marrow. Within 24 h after irradiation, mice received bone marrow transplant from 6-week-old GFP transgenic mice through tail vein as a 200 μl cell suspension containing 0.7–1.0 × 10⁷ cells. 6 weeks later, mice were subjected to IR.

Bone marrow was aseptically harvested from tibias, femurs and humeri from 9-week-old mice, erythrocytes lysed (BD Pharm Lyse buffer) and cells incubated with Fc-block followed by incubation with fluorochrome-conjugated antibodies against B220, CD4, CD8, CD11b, Ly6G, NK1.1, Siglec F, Ter119, c-Kit and Sca-1 (BD Biosciences, eBioscience, Biolegend), as previously described [38 (link)]. Cell suspensions were sorted by fluorescence-activated cell sorting (FACS) to obtain the Lin^-Sca-1⁺c-kit ⁺ (LSK) population. Two-month-old B6.CD45.1 mice received a single 1000-cGy γ-irradiation dose using a Cs-137-based Gammacell-40 irradiator (Nordion). After 12 hours, 5 × 10⁴ LSK cells were intravenously injected from: Casp8^flox/flox, Cre^CD11cCasp8^flox/flox, RIPK3^–/–Cre^CD11cCasp8^flox/flox, a mixture of Casp8^flox/flox plus B6.CD45.1/2, Cre^CD11cCasp8^flox/flox plus B6.CD45.1/2 or RIPK3^–/–Cre^CD11cCasp8^flox/flox plus B6.CD45.1/2 (1:1 ratio). Chimeric mice were maintained on Trimetoprim/Sulfamethoxazole (40 mg/5 mg, respectively; Hi-Tech Pharmacal) diluted in autoclaved water (2 mL antibiotics/500 mL water) and phenotyped 2 months post transfer.

SWT (XSD Pharmacy Company, lot Number 120306, Chengdu, China), was administered using two different regimens. In one regimen mice were treated with SWT for 7 days prior to irradiation until one day before irradiation. A non-irradiated group and an irradiated group were treated with PBS at the same times that SWT treatments were performed to serve as controls. In the second group, mice were treated with SWT starting seven days prior to irradiation and continued after irradiation until one day before blood sample collection. Also, a non-irradiated group and an irradiated group were treated with PBS at the same time that SWT treatments were performed to serve as controls. For both regimes, blood samples were collected on days 4, 10, 13, 16, 18 and 20 post-irradiation. SWT and PBS were administered by daily oral gavages for the time periods indicated, in a total volume of 200 μl.
For irradiation, mice were exposed to a single dose, 2 Gy, of total body irradiation administered at a dose rate of 0.41 Gy/min using a ¹³⁷Cs source (Gammacell 40 irradiator, Nordion, Ottawa, ON, Canada).

Cells were γ‐irradiated in a 137Cs gamma‐ray source (Gammacell 40 irradiator, MDS Nordion) with indicated dose, and chromosome fragmentation was determined by comet assay. Briefly, during irradiation all the cells are kept in ice to stop the DNA repair process. Thereafter, cells were transferred to 37°C to allow DNA repair and then harvested at indicated time points for analysis. 1 × 10⁵ cells/ml in cold PBS were resuspended in 1% low‐melting agarose at 40°C at a ratio of 1:3 vol/vol and pipetted onto a CometSlide. Slides were then immersed in prechilled lysis buffer (1.2 M NaCl, 100 mM EDTA, 0.1% sodium lauroyl sarcosinate, 0.26 M NaOH, pH > 13) for overnight (18–20 h) lysis at 4°C in the dark. Slides were then carefully removed and submerged in room temperature rinse buffer (0.03 M NaOH and 2 mM EDTA, pH > 12) for 20 min in the dark. This washing step was done 2 times.
Slides were transferred to a horizontal electrophoresis chamber containing rinse buffer and separated for 25 min at voltage 0.6 V/cm. Finally, slides were washed with distilled water and stained with 10 μg/ml propidium iodide and analyzed by fluorescence microscopy. Twenty fields with about 200 cells in each sample were evaluated and quantified by the Fiji software to determine the tail length (tail moment).