PBMCs were isolated by density gradient centrifugation using Biocoll (Biochrom, Cambridge, United Kingdom), and then further washed to improve platelet removal

PBMCs were isolated by density gradient centrifugation using Biocoll (Biochrom, Cambridge, United Kingdom), and then further washed to improve platelet removal. When stimulated with whole tumor cells lysates, both MoDCs expressed similar levels of maturation and co-stimulatory markers. However, when cultured with autologous T cells, NS11394 MACS_MoDCs induced significantly higher IFN- secretion than EasySep_MoDCs, indicating a stronger induction of Th1 cell response profile. Concordantly, T cells induced by MACS_MoDCs also showed a higher release of cytotoxic granules when in contact with tumor cells. Conclusions Overall, both the MACS and the EasySep isolation immunomagnetic technologies provide monocytes that differentiate into viable and functional MoDCs. In our experimental settings, resting EasySep_MoDCs showed a higher basal level of maturation but show less responsivity to stimuli. On the other hand, MACS_MoDCs, when stimulated with tumor antigens, showed better ability to stimulate Th1 responses and to induce T cell cytotoxicity against tumor cells. Thus, monocyte isolation techniques crucially affect MoDCs function and, therefore, should be carefully selected to obtain the desired functionality. lipopolysaccharide (LPS) was from Sigma-Aldrich (St. Louis, Mo, USA). Cell Counting and Viability Examination Cells were counted using a Neubauer chamber, following staining with trypan blue. Cell viability was also evaluated by flow cytometry, after staining with 7-Aminoactinomycin D (7AAD) (BD Biosciences, NJ, USA). Isolation of NS11394 Peripheral Blood Mononuclear Cells Peripheral blood mononuclear cells (PBMCs) were obtained from leuko-platelet concentrates from healthy donors, from the Portuguese Blood and Transplantation Institute (Instituto Portugus do Sangue e da Transplanta??o – IPST); and approval from the institutional ethical committee was previously obtained. PBMCs were isolated by density gradient centrifugation NS11394 using Biocoll (Biochrom, Cambridge, United Kingdom), and then further washed to improve platelet removal. Each PBMCs sample was divided and processed in parallel with both immunomagnetic separation kits, as described below. HLA typing was performed and only donors with an HLA-A*02:01 profile were selected for the cytotoxicity assays. Isolation of CD14+ Monocytes Using CD14 MicroBeads from Miltenyi C MACS Technology Monocyte isolation using the positive immunomagnetic selection kit from Miltenyi Biotec was performed according to the manufacturers instructions and as described [11, 12]. PBMCs were resuspended in phosphate-buffered saline (PBS) buffer, pH?7.2, containing 0.5% bovine NS11394 serum albumin (BSA), and 2?mM ethylenediamine tetraacetic acid (EDTA); and incubated with CD14 microbeads (20?L per 107 cells) during 15?min at 4?C. The cell suspension was loaded onto an LS magnetic column (Miltenyi Biotec) placed in the magnetic field Rabbit Polyclonal to ELOVL5 of a MACS Separator (MIDIMACS) and rinsed three times with buffer. At this point, the CD14-positively labeled cells were retained in the magnetic field, while the negative cells were eluted. The column was then removed from the magnetic field, followed by the elution of the CD14+ fraction. Cell fractions were washed: CD14 cells were cultured and CD14neg (CD14) cells were frozen. Isolation of CD14+ Monocytes Using EasySep Human CD14 Selection Kit from StemCell C EasySep Technology Monocyte isolation using NS11394 the positive selection kit from StemCell Technologies (Vancouver, BC, Canada) was performed according to the manufacturers instructions. Briefly, PBMCs were resuspended in PBS with 2% FBS and 1?mM EDTA and magnetically labeled in a two-step process. Firstly, PBMCs were incubated for 15?min at room temperature with Positive Selection Cocktail, tetrameric antibodies complexes (TAC) that recognize both CD14, and dextran. Then, dextran-coated EasySep Magnetic Nanoparticles were added and incubated 10?min at room temperature to allow them to bind to the TAC particles. The tube with the mixture was placed into an EasySep Magnet and incubated for 5?min, after which it was inverted to pour off the supernatant. At this point, magnetically labeled CD14+ cells remain inside the tube and were resuspended in buffer. The supernatant was re-incubated twice with the magnet and the remaining CD14+ cells were harvested and cultured and the CD14? cells were frozen. Generation and Maturation of MoDCs Monocytes isolated by either one of the previously described methods were resuspended at a density of 1 1 106 cells/mL in culture media supplemented with 750?U/mL IL-4 and 1000?U/mL GM-CSF. The cell culture was plated in 6-well tissue culture plates and incubated for 7?days. Every 2 days, half of the culture media was replaced by fresh media supplemented with cytokines. For the maturation of MoDCs, culture media was supplemented.