B6.129s4 il2rgtm1wjl j mice

Bone marrow cells were harvested from 6 week old B6.129S4-Il2rg^tm1Wjl/J mice (Jackson Laboratory, Bar Harbor, Maine). Cells were subjected to red cell lysis in NH₄Cl solution (0.8% NH₄Cl with 0.1 mM EDTA). Lineage-cells were enriched using a Lineage Cell Depletion Kit according to the manufacturer’s protocol (Miltenyi Biotech, Germany). Cells were pre-stimulated at 37°C in DMEM with 15% FCS, 15% WEHI-3B, murine IL-3 (7 ng/mL), IL-6 (12 ng/mL), and SCF (56 ng/mL) (Stem Cell Technologies, Canada). JAK3 mutants and IL2Rγ retroviruses were generated as described above. After 24 hrs, the cells were spinoculated into 6-well plates with viral supernatant in pre-stimulation medium in the presence of 5 μg/mL polybrene and HEPES at 2,500 rpm for 90 mins. This was repeated at 48 hrs. Cells expressing JAK3 and IL2Rγ were doubly sorted by FACS, gated on GFP (JAK3) and anti-human IL2Rγ-PE (BioLegend). 1×10⁴ cells per 35 mm dish were plated in triplicate in methylcellulose medium in the absence of cytokines (MethoCult M3234, Stem Cell Technologies). Colonies were scored following 8 days of incubation at 37°C in 5% CO₂.

C57BL6/J (Jackson 000664), BALB/cJ (Jackson 000651), MMTV-PyMT (FVB/N-Tg(MMTV-PyVT)634Mul/J, Jackson 002374), Rag2^−/− (B6(Cg)-Rag2^tm1.1Cgn/J, Jackson 008449), TNFR KO (B6.129S-Tnfrsf1a^tm1ImxTnfrsf1b^tm1Imx/J, Jackson 003243), C3^−/− (B6.129S4-C3^tm1Crr/J, Jackson 029661), and Ncf1^−/− (B6N.129S2-Ncf1^tm1Shl/J, Jackson 027331) mice were purchased from Jackson Laboratory. Fcer1g^−/− mice (B6.129P2-Fcer1g^tm1Rav N12, Taconic 583) were purchased from Taconic. Rag2^−/−Il2rg^−/− mice were generated by crossing B6(Cg)-Rag2^tm1.1Cgn/J mice (Jackson 008449) with B6.129S4-Il2rg^tm1Wjl/J mice (Jackson 003174). Cfp^−/− mice^{67 (link),74 (link)} and C6^−/− mice^{75 (link)} were generated as previously described. 8–12 week old female mice were used, and mice of different experimental groups and genotypes were cohoused during all experiments, with the exception of immunocompromised Rag2^−/− mice. Mice were randomly assigned to experimental groups. All animal studies were performed in accordance with the Stanford University Institutional Animal Care and Use Committee under protocol APLAC-17466. All mice were housed in an American Association for the Accreditation of Laboratory Animal Care-accredited animal facility and maintained in specific pathogen-free conditions.