(B) IL-1beta release from PRRSV and LPS-treated PBMC cultures
PRRSV strains	Virus only (pg/ml)	Virus + LPS (pg/ml)	Delta: (virus + LPS) – LPS only
BS773	2,655	>8,000	≥674
21377	6,442	7,145	−181
433/5	6,195	>8,000	≥674
BS114	1,892	2,786	−4,540
Controls			pg/ml
LPS only			7,326
LPS + ATP			6,040
Medium only			4,315

Aliquots of PBMCs of six PRRS-naive pigs in liquid nitrogen were thawed and screened for spontaneous IL-1beta release. Then, a further aliquot of the PBMCs showing the lowest level of release was thawed, and cells were grown in 0.5 ml/well of RPMI 1640 medium + 10% heat-inactivated FCS and LPS (1 μg/ml final) at 37°C, 5% CO₂, in agarose-treated, 48-well microtiter plates to induce detachment-induced cell death (anoikis). After 4 h, different PRRSV strains at MOI 2 or ATP (4.9 mM final, inflammasome control) were added, and PBMCs were further grown for another 18 h. Then, 150 μl/well of supernatant were carefully removed to investigate the IL-1 beta response, whereas the cells were employed in a FAM-FLICA flow cytometry assay for caspase-1. Singlet cells were analyzed in a quadrant cytogram for green (caspase-1) and red (PI) fluorescence as shown in Figure 2. The two experiments were carried out on four PRRSV strains. (A) % live, caspase 1-positive cells and the corresponding dead ones (late apoptotic and necrotic) are reported. For each result, a delta value is reported as the % difference between (virus + LPS) and (virus only). For each delta value, the corresponding P value is shown in the next column on the right. NS, not significant. The significant increase of live, caspase 1-positive cells in attenuated PRRSV-stimulated PBMCs is highlighted with an asterisk. (B) The concentrations of IL-1beta in the supernatants and the corresponding Delta values (virus + LPS) – (LPS only) are reported for each experimental condition in terms of pg/ml.