Anti human il2rγ pe

Ba/F3 cells were cultured in standard media containing RPMI 1640 media, 10% fetal bovine serum (FBS), L-glutamine, penicillin-streptomycin (Life Technologies, Grand Island, NY) containing IL-3 from WEHI conditioned media. JAK3-GFP mutant and IL2Rγ retrovirus was generated by transfecting 293T/17 cells with EcoPac helper plasmid (a kind gift of Dr. Rick Van Etten, Tufts University, Boston, MA) and MSCV retroviral constructs using Fugene (Roche Diagnostics Corp., Indianapolis, IN). For infection, 1 mL of viral supernatant was used to infect 1×10⁶ cell using two rounds of spinoculation in the presence of 5ug/mL polybrene and HEPES at 2,500 rpm for 90 mins. Cells were cultured for several days, followed by sorting of double positive cells using FACSAriaII (BD Bioscience, San Jose, CA) for JAK3 and IL2Rγ expression gated on GFP and anti-human IL2Rγ-PE (BioLegend, San Diego, CA), respectively. Equal density of stable cell lines plated in medium lacking WEHI-conditioned medium (source of IL-3), and total viable cells were counted every other day for three weeks.

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.