Round bottomed 96 well plates

After seven days of cultivation, moDCs were seeded into round-bottomed 96-well plates (Greiner Bio-One) at 2 × 10⁵ cells per well and infected with PRRSV-1 or PRRSV-2 strains at an MOI of 0.1 at 37 °C in 5% CO₂. After two hours, the inoculum was removed and cells were washed twice with CM. Separate microcultures with mock-infected moDCs were included in each experiment. Cultivation of moDCs was continued until 12, 24 and 48 hours post-inoculation (hpi). At these time points moDCs were harvested and analyzed for PRRSV-N-protein expression (see below for details). In addition, moDCs were infected with different MOI (0.1, 0.02 and 0.004) and harvested after 24 hpi. Harvested moDCs were labelled with anti-CD172a mAbs (see Table 1 for details on staining strategy) and isotype-specific secondary antibodies in combination with Live/Dead® Fixable Near-IR during a second incubation step. Subsequently, moDCs were fixed and permeabilized by the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA, USA) according to manufacturer’s instructions. Thereafter, cells were incubated with anti-PRRSV-N-protein mAbs (clone P10/b1) [29 (link)]. This antibody had been purified and conjugated to Alexa488 as described elsewhere [30 (link)].

To generate MTT spheroids, 5 × 10³ cells/well in cell-repellent surface, round bottomed 96-well plates (Greiner Bio-one, Kremsmünster, Austria), were centrifuged at 220× g at room temperature for 10 min. This procedure generated spheroids with homogeneous size and geometry. After 48 h spheroids showed a round morphology with clear margins and used for further experiments as T0 [27 (link)]. To assess spheroids growth, culture medium was replaced with fresh medium with or without 4 mM of metformin. Images of the spheroids were acquired at different time points (T0, 2, 3, and 6 days), and spheroid diameters were measured with ImageJ software [34 (link)].

The mononuclear cell fraction was isolated by density centrifugation of EDTA anticoagulated blood, diluted 1 : 1 in pyrogen-free saline, over Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden). Isolated cells were washed twice in saline and resuspended in culture medium (RPMI, Invitrogen, Carlsbad, California, USA) supplemented with 10 μg/mL gentamicin, 10 mM L-glutamine, and 10 mM pyruvate. Cell counts were performed in a Coulter counter (Coulter Electronics). A total of 5 × 10⁵ mononuclear cells in 100 μL were added to round-bottomed 96-well plates (Greiner) with RPMI, sonicated Mycobacterium tuberculosis (MTB) (1 μg/mL end protein concentration, strain H37Rv), Escherichia coli lipopolysaccharide (LPS; 1 ng/mL; Sigma-Aldrich, St. Louis, MO, USA), heat-killed Staphylococcus aureus (1 × 10⁶ microorganisms/mL, clinical isolate), or heat-killed Candida albicans (1 × 10⁶ microorganisms/mL, strain UC820). After 24 h (for determination of TNF-α, IL-1β, and IL-6) or 48 h (for determination of IFN-γ and IL-10) of incubation, plates were centrifuged and supernatants were stored at −20°C until analysis. Cytokines were measured batchwise using commercially available ELISAs (R&D Systems, MN, USA, and Sanquin, Amsterdam, Netherlands) according to the protocols supplied by the manufacturers.