Rhil 3

Frozen human mobilized peripheral blood (mPB) CD34⁺ cells were purchased from AllCells. Upon receipt, mPB CD34⁺ cells were thawed and counted prior to assessment of viability via trypan blue dye exclusion. For transduction, mPB CD34⁺ cells were re-plated at a cell density of 1 × 10⁶ cells/mL in RN-coated plates in stem cell growth media (SCGM) containing 300 ng/mL recombinant human Flt-3 Ligand (rhFlt-3L), 300 ng/mL recombinant human stem cell factor (rhSCF), 100 ng/mL recombinant human thrombopoietin (rhTPO), and 60 ng/mL recombinant human IL-3 (rhIL-3) and stimulated overnight at 37°C/5% CO₂. SCGM, rhFlt-3L, rhSCF, rhTPO, and rhIL-3 were all purchased from CellGenix.

Isolated DC-like subsets were resuspended in X-VIVO 15 (Cambrex) containing 2% pooled human serum (Sanquin). pDCs were supplemented with 10 ng/ml recombinant human IL-3 (rhIL-3; Cellgenix). Both cell types were stimulated for 4 hr or overnight with 4 μg/ml R848, followed by 45-min stimulation with 5 mM ATP (Sigma). Culture supernatant was taken for ELISA.