Hla dr buv395

Flow cytometry analysis of THP-1 cells and total blood was performed using the following antibodies. Conjugated anti-human SR-B1 APC (Miltenyi Biotec 130-111-237), CD235a APC Cy7 (BioLegend 3,49,115), CD45 BUV805 (BD Biosciences 6,12,892), CD56 BUV 737 (BD Biosciences 6,12,767), CD11c PeCy7 (BioLegend 3,37,216), CD11b PE (eBioscience 12-0118-41), CD3 PerCP Cy5.5 (BD Biosciences 5,60,835), CD19 BV786 (BioLegend 3,02,239), HLA-DR BUV 395 (BD Biosciences 7,40,302), CD14 BV605 (BioLegend 3,01,834). Labeling of mouse blood was done using the following conjugated antibodies: CD45.2 PE (eBioscience 12-0454–82), CD115 PerCPeF710 (eBioscience 46-1,152-82), CD11b BV650 (eBioscience 48-0112–20), Ly6G BV510 (BD Biosciences 5,63,402), CD3 APC-eF780 (BioLegend 47-0032–82). Fc receptors were blocked using FCR blocking reagent (Myltenyi Biotec). Surface membrane staining was performed in PBS FBS 2%. The Zombie UV fixable viability dye (BioLegend 4,23,107) was used to exclude dead cells. Samples were acquired on a Cytoflex (Beckman Coulter) and analyzed with FlowJo 10 (BD Biosciences).

Peripheral blood mononuclear cells (PBMCs) from human healthy donors were thawed from ImmunXperts SA (Belgium) biobank. Monocytes were isolated from PBMCs using a MACS magnetic separation column CD14 MicroBeads (Miltenyi) and purity was evaluated by CD14 FACS staining (Fortessa). Cells were then resuspended at a cellular density of 10⁶ cells/mL and plated into a 24-well tissue culture microplate (1mL per well) in CellGenix DC medium (CellGenix, Cat.N° 20801-0500) added with Gentamycin, IL-4 (Miltenyi, 130-093-866) and GM-CSF (Miltenyi, 130-093-922) for 5 days. At day 5, cells were stained for FACS analysis with several DC activation markers to assess their immature dendritic cell (iDC) state: CD14-FITC (Miltenyi, 130-110-518), CD40-BV510 (BD Biosciences, 563456), CD80-BV421 (BD Biosciences, 564160), CD83-PE-Vio 770 (Miltenyi, 130-110-505), CD86-APC (Miltenyi, 130-116-161), CD209-PE (Miltenyi, 130-117-706), and HLA-DR-BUV395 (BD Biosciences, 564040). On the same day, respective antigens (10µg/mL) were added to the cell culture for 48h. At day 7, cells were stained for FACS analysis with same markers to assess their mature state. LPS (Sigma, L4391-1MG) (1µg/mL), and TNF (Miltenyi, 130-094-024) (800U/mL), with IL-1b (Miltenyi, 130-093-898) (150U/mL), were used as positive control.

PBMCs were stained with CD45RA-FITC (BioLegend), CD8- Percp cy5.5 (eBioscience), β7-APC (BD Biosciences), CD127-APCef780 (eBioscience), CD25-BV421(BD Biosciences), CD4-BV650 (BD Biosciences), CCR6-BV711 (BD Biosciences), CD3-BV785 (BD Biosciences), α4-PE (BD Biosciences), CCR5-PE-CF549(BD Biosciences), CCR7-Pe-cy7 (BD Biosciences), HLA-DR-BUV395 (BD Biosciences), CD69-BUV737 (BD Biosciences) and Live/Dead Aqua (Invitrogen). Cells were enumerated using a BD LSR Fortessa X20 flow cytometer (BD Systems) and analyzed with FlowJo 10.4.1 software (TreeStar, Ashland, OR) by the same researcher for consistency.