20 fortessa sorp flow cytometer

CD14⁺ monocytes were isolated from PBMCs by positive selection using anti-CD14 conjugated magnetics beads according to the manufacturer’s instructions. Monocytes were then cultured for 5 days in the presence of 500 U/mL IL-4 and 800 U/mL GM-CSF for the generation of immature moDCs. Peripheral blood lymphocytes (PBLs) from healthy donors were labeled with 5 μM violet tracer, according to the manufacturer’s instructions and co-cultured with allogeneic ApoEVs and ApoEVs-HM (200 μg/mL) loaded and LPS-stimulated (10 ng/mL) (24 h) moDCs in complete IMDM medium for 7 days. Control moDCs were stimulated with 5 ng/mL SEB or 5 μg/mL PHA-L. At day 7, cells were harvested and labeled with anti-CD4 and anti-CD8. Proliferation was determined using flow cytometry (×20 Fortessa SORP flow cytometer, BD Biosciences) and analyzed with FlowJo V10 software (Ashland, OR, USA).

ApoEVs were labeled with 0.5–1 μM DiD for 15 min at 37 °C. After labeling, vesicles were washed twice in PBS and centrifuged at 10,000× g for 30 min. Blocking DC-SIGN and MR was done by pre-treating the DCs with AZN-D1 (20 μg/mL), anti-MR (CD206) (40 μg/mL), or isotype control (40 μg/mL) for 20–30 min at 37 °C. Immature or maturing (10 µg/mL MPLA) moDCs (10 × 10⁴ per time point) were loaded with 50 μg/mL DiD labeled ApoEVs or ApoEVs-HM for 45 min on ice to allow binding and thereafter transferred to 37 °C. At different time points (0, 30 and 60 min) cells were washed, and subsequently fixed with 1% PFA. The cells were analyzed by flow cytometry (×20 Fortessa SORP flow cytometer, BD Biosciences).

Immature moDCs were seeded in a 96-well U-bottom plate (Greiner) at a concentration of 50 × 10³ cells per well and loaded with the different vesicles (2 donors 200 µg/mL and 2 donors 100 µg/mL or LPS (10 ng/mL)). After 3 h, moDCs were washed in PBS and cultured o/n. MoDCs were stained for the expression of DC maturation markers CD80, CD86, and HLA-DR for 30 min. Marker expression was measured by flow cytometry (×20 Fortessa SORP flow cytometer, BD Biosciences).