Hematopoietic Stem Cell Transplantation

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.