Lymphocyte separating medium

The peripheral blood mononuclear cells (PBMCs) were isolated from 30 ml of HLA-A2⁺ healthy donor blood with written consent under Siriraj Institutional Review Board ethical approval (COA no. Si 580/2018) by density centrifugation at 800 × g for 20 min at RT in lymphocyte separating medium (Corning Life Sciences). The monocytes were isolated and incubated for 1 h at 37°C. The non-adherence cells were collected and cryopreserved in human AB serum (MilliporeSigma) containing 10% dimethyl sulfoxide until use. The monocytes were transduced with IRFP-SmartDC or MSLN-SmartDC at 75 multiplicity of infection (MOI) together with 10 µg/ml protamine sulfate in AIM-V medium (Invitrogen; Thermo Fisher Scientific, Inc.). On day 5 post-transduction, 1 µg/ml of recombinant human RPS3 (cat. no. NBP2-90977; Novus Biologicals, LLC) was added. Monocytes were cultured in 100 ng/ml of rhGM-CSF (cat. no. 11343125; ImmunoTools GmbH) and 50 ng/ml of rhIL-4 (cat. no. 11340045; ImmunoTools GmbH) for five days and treated with 100 ng/ml of rhIFN-γ (cat. no. 11343536; ImmunoTools GmbH) and rhTNF-α (cat. no. 11343015; ImmunoTools GmbH) for additional 48 h served as positive control or conventional DC (conv. DC). All DCs were harvested on day 7 to check the activated characters.

PBMCs from HLA-A*02–positive, were isolated with Lymphocyte separating medium (Corning). This present study was approved by Siriraj Institutional Review Board (COA no. Si 580/2018) and conducted in accordance with the Declaration of Helsinki. The written informed consent was obtained from all healthy donors. On day 0, PBMCs (1×10⁶/well) were plated in 12-well plates (Corning). For ConvDCs growth, the adherent monocytes were cultured in AIM-V media (Invitrogen Corporation) with GM-CSF (50 ng/m, ImmunoTools GmbH) and IL-4 (25 ng/mL, ImmunoTools GmbH; ref. 28 ). Transduction was carried out on day 5 of culture with LV (100 multiplicity of infection) and protamine sulfate (10 µg/mL) added to adherent monocytes and incubated for 16 hours at 37°C, the cells were replaced with AIM-V media to activate T cells. The immature DCs were cultured in AIM-V media with IFNα (50 ng/mL, ImmunoTools GmbH), IFNγ (50 ng/mL, ImmunoTools GmbH), and recombinant human RPS3 (1 µg/mL, Novus Biological) for 2 days to induce mature DCs. At day 7 of culture, the phenotypic analysis of DCs and SmartDCs was compared with monocytes performed by the CytoFLEX Flow cytometer (Beckman Coulter) and analyzed by FlowJo VX software (Tree star, Inc.).