Qbsf 60 medium

MSC cultures at 60% confluence were incubated overnight with supernatants containing lentiviral particles encoding HLA-G1 diluted in serum-free QBSF-60 medium (Quality Biological, Gaithersburg, MD) and 8 μg/ml protamine sulfate (Calbiochem, San Diego, CA). Transduction was performed at an MOI that yielded similar levels of HLA-G1-expressing MSC for all gene delivery systems. After transduction, cells were washed, and media was changed to MSCGM (Lonza). Stably transduced MSC with HLA-G1 (LvMSC-HLA-G1) were analyzed for transgene expression using flow cytometry.

RS4;11, Jurkat, CEM, NALM6 cell lines were all obtained from commercial suppliers and used within 3 months of culture following validation by short tandem repeat analysis. Cell lines were maintained in RPMI supplemented with 10% heat inactivated fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine (Life Technologies, Carlsbad, CA). ALL cell lines were validated by Short Tandem Repeat analysis, verified mycoplasma-free and cultured for <3 months. Xenograft cells were retrieved from cryostorage and resuspended in QBSF-60 medium (Quality Biological, Gaithersburg, MD) supplemented with 20 ng/ml Flt-3 ligand, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. Prior to treatment, cells were plated in 96-well plates (100 μl/well) at a density previously optimized and were equilibrated overnight at 37°C with 5% CO₂. Cells were then treated with 10-fold serial dilutions of RG7112 (10 μM – 1 pM) for 48 h, at which point AlamarBlue reagent [0.6 mM Resazurin, 0.07 mM Methylene Blue, 1 mM potassium hexacyanoferrate (III), 1 mM potassium hexacyanoferrate (II) trihydrate] was added. Fluorescence was measured at 0 and 6 h following addition of AlamarBlue using a fluorescent plate reader (VICTOR³™, PerkinElmer, MA) with excitation at 560 nm and emission at 590 nm. Data are expressed as a percentage of untreated controls.

Xenograft cells were retrieved from cryostorage and resuspended in QBSF-60 medium (Quality Biological, Gaithersburg, MD) supplemented with Flt-3 ligand (20 ng/mL), penicillin (100 U/mL), streptomycin (100 μg/mL) and L-glutamine (2 mM). Viability was determined by trypan blue exclusion. Prior to treatment, cells were plated in 96-well plates (100 μL/well) at a density previously optimized (3–6 × 10⁶/mL) and equilibrated overnight at 37°C in 5% CO₂. Cells were exposed to 10-fold serial dilutions of AZD1480 or selumetinib (AstraZeneca, North Ryde, NSW) (10⁻⁶ to 10⁻¹² M) for 72 h, following which AlamarBlue reagent (Life Technologies, Carlsbad, CA) was added and fluorescence was measured at 0, 6 and 24 h using a fluorescent plate reader (VICTOR^3™, PerkinElmer, Waltham, MA) (excitation 560 nm, emission 590 nm). For fixed ratio combination assays, cells were treated with AZD1480 and selumetinib, alone or in combination, at 4, 2, 1, 0.5, 0.25 and 0.1 μM. The combination drug effect was assessed using Calcusyn software (Version 2.0, Biosoft, Cambridge, UK) to calculate Combination Indices (CIs) indicative of synergy (CI<1), additivity (CI=1) or antagonism (CI>1).