Lymphoprep gradient

Peripheral blood lymphocytes were enriched using a lymphoprep gradient (Axis Shield, Oslo, Norway) as described (11 (link)), and monocytes were purified with magnetic beads (Stemcell Technologies, Köln, Germany). Purity was assessed on the basis of CD14/CD16 staining by flow cytometry using a FACSCanto II device (BD Biosciences, Heidelberg, Germany), and routinely >95% (Figure 1). Monocytes were analyzed directly or cultured for 3 h in RPMI 1640 medium supplemented with 0.5% fatty acid-free BSA under serum-starved conditions in the presence or absence of 10⁻⁶ M MP. One portion of the cells was used for RNA isolation and surface marker analyses and the other portion served to assess the migratory capacity.

Heparinized syringes were used to obtain peripheral blood samples (20 mL) by venipuncture. Layering over the surface of a discontinuous LymphoPrep gradient (Axis-Shield, Oslo, Norway) and subsequent centrifugation were utilized to isolate PBMCs from each blood sample. A protocol involving two washings of isolated PBMCs with RPMI 1640 (GIBCO™, Thermo Fisher Scientific, Waltham, MA, USA) was applied afterwards. Cells were then enumerated using a Neubauer hemocytometer. Moreover, cell viability was assessed for each sample by trypan blue exclusion dye and routinely surpassed 95%. A cell cryoprotective solution consisting of 10% DMSO, 30% RPMI 1640, and 60% fetal calf serum (FCS) was produced. PBMCs were then re-suspended in that medium and aliquoted into cryotubes (Corning™, Thermo Fisher Scientific, Waltham, MA, USA). Aliquots were kept at − 80 °C for 24 h, and then were stored in liquid nitrogen containers until further use.

Human PBMCs were purified from citrated blood using Lymphoprep gradient (AXIS-SHIELD), according to the manufacturer instructions. Equal volumes of PBMCs suspensions (5x10⁶ cells/mL) and leptospires (2x10⁷/mL) or bacteria culture supernatants were mixed and incubated at 37°C for 2 h. Buffer alone or EMJH were used as controls. The PBMCs were pelleted by centrifugation and resuspended in 600 μL normal plasma or FVII-deficient plasma diluted 50% in HEPES-ZnCl₂. Normal plasma was used to verify the general status of the coagulation, while FVII-deficient plasma as a control to exclude the activity of the TF/extrinsic pathway of coagulation. The recalcification clotting times were measured by addition of 50 μL 30 mM CaCl₂ to 100 μL of sample. Alternatively, the aPTTs were measured in order to verify the status of the intrinsic (contact system) and common pathways of coagulation. aPTTs were obtained by addition of 50 μL aPTT reagent to 50 μL of sample followed by 200 s incubation and addition of 50 μL 30 mM CaCl₂.