Rpmi 1640

Human buffy coats and serum were from the blood bank at St. Olavs Hospital (Trondheim, Norway), with approval by the Regional Committee for Medical and Health Research Ethics (REC) in Central Norway. Primary human monocytes were isolated from the buffy coat by adherence, as previously described (56 (link)). In brief, freshly prepared buffy coats (St. Olavs Hospital) were diluted by 100 ml of PBS and applied on top of Lymphoprep (Axis-Shield) according to the manufacturer’s instructions. PBMCs were collected and washed by HBSS (Sigma-Aldrich, Merck) four times with low-speed centrifugation (150–200g). Cells were counted using Z2 Coulter particle count and size analyzer (Beckman Coulter) on program B, resuspended in RPMI 1640 (Sigma-Aldrich, Merck) supplemented with 5% of pooled human serum at a concentration of 8 × 10⁶ per ml, and seeded to six-well (1 ml per well) or 24-well (0.5 ml per well) cell culture dishes. After a 45-min incubation allowing surface adherence of monocytes, the dishes were washed three times by HBSS to remove nonadherent cells. Monocytes were kept overnight in RPMI1640 (Sigma-Aldrich, Merck), supplemented with 30% of pooled human serum, followed by media change to RPMI1640 with 10% human serum before experimental procedures.

Vaccinated mouse spleens were isolated to test stimulating cytokine productions of splenocytes at 70 days post-vaccination (dpv), and unvaccinated mouse spleens were also collected to use as controls. In brief, the splenocytes were washed three times with RPMI 1640 (Sigma, United States), and hemolyzed in a lysing buffer (0.83% NH₄Cl and 0.01 M Tris-HCl, pH 7.2) for 5 min, then washed with RPMI 1640. The viability of the splenocytes was determined by trypan blue staining. A total of 3 × 10⁵ viable splenocytes each well of 96-well cell culture plates were plated and cultured in RPMI 1640 supplemented with 20% FBS maintained 24 h. The final concentration of 50 μg/ml of T. gondii soluble antigens (TSA) of PLK parasites were used to stimulate cytokine productions for 3 days. Then supernatants from each well were harvested for cytokine level measurements, as above. For negative and positive controls, the same number of splenocytes was also plated into 96-well cell culture plates at the same time and stimulated with RPMI 1640 with 20% FBS only or 5 μg/ml concanavalin A (Sigma, United States) for 3 days, respectively. Each spleen-harvested splenocytes were plated in at least three wells for TSA, negative and positive control, and each supernatant sample was tested three times.

Peritoneal exudate cell and PLEC were isolated by flushing the peritoneal and pleural cavities with RPMI 1640 (Sigma), respectively. Pericardium and mediastinum were enzymatically digested with 1 mg ml⁻¹ Collagenase D (Roche) for 35 min at 37 °C in RPMI 1640 containing 1% fetal bovine serum (Sigma). PLEC, lymph node cells or FALC cells, were cultured overnight and supernatant used for ELISA. Equivalent weights of lung, whole mediastinum, whole omentum and GAT were cultured for 1 h before determination of IL-33 in supernatant by ELISA. For analysis of intracellular IL-5 and IL-33, pericardium and PLEC cells were used directly ex vivo or rested overnight in RPMI 1640 (Sigma) containing 10% fetal bovine serum (Sigma), 50 U ml⁻¹ Penicillin (Sigma) 50 μg ml⁻¹ Streptomycin (Sigma), 2 mM L-glutamine (Sigma), cells were then stimulated for 4 h at 37 °C with 50 ng ml⁻¹ phorbol-myristate acetate (Sigma) and 1 μg ml⁻¹ Ionomycin (Sigma) including 1 × Brefeldin A (eBioscience) for the final 3 h of incubation.

The AmB MBEC is the lowest concentration, recorded in mg/L, of the drug able to reduce the biofilm cell population to at least 2 Log₁₀ CFU per cm². For this determination, standardized cell suspensions (200 μL) were placed into selected wells of 96-well polystyrene microtiter plates (Orange Scientific, Braine-l’Alleud, Belgium). RPMI 1640 (Sigma-Aldrich, St. Louis, MO, USA) was used without cells, but with antifungal agent, as a negative control. As positive control, cell suspensions were tested without antifungal agent. At 24 h, 100 μL of RPMI 1640 (Sigma-Aldrich, St. Louis, MO, USA) was removed, and an equal volume of fresh RPMI 1640 plus the respective antifungal concentration was added (2, 3, 4, 8 mg/L, 2× concentrated). The plates were incubated at 37 °C for another 24 h, a total of 48 h at 120 rpm. The number of cultivable cells on biofilms was determined by the enumeration of CFUs. For this, after the period of biofilm formation, all medium was aspired, and the biofilms were washed once with 200 μL of PBS to remove non-adherent cells. Then, biofilms were scraped from the wells, and the suspensions were vigorously vortexed for 2 min to disaggregate the cells from the matrix. Serial decimal dilutions in PBS were plated on SDA and incubated for 24 h at 37 °C. The results were calculated as a total of CFUs per unit area (Log₁₀ CFUs/cm²), and presented by mg/L [101 (link)].

Murine gonadal AT were enzymatically digested with 1 mg/ml Collagenase D (Roche) for 35 min at 37 °C in RPMI 1640 (Sigma) containing 1% fetal bovine serum (Sigma). Peritoneal exudate cells were isolated by flushing murine peritoneal cavities with RPMI 1640 (Sigma). The liver was perfused before dissection with 5 ml of RPMI 1640 (Sigma) injected through the portal vein. The tissue was cut into small pieces and homogenized using the gentleMACS dissociator (Miltenyi) in buffer containing Collagenase 2 (Sigma 0.425 mg/ml), Collagenase D (Roche 0.625 mg/ml), Dispase (Gibco 1 mg/ml), and DNase (Roche 30 µg/ml). After 20-min incubation at 37 °C, the tissue was homogenized further using the dissociator. Red blood cells were lysed using red blood cell lysis buffer (Sigma).