Victor3 1420 plate reader

Monocytes isolated based on plastic adherence as described above were plated at 5 x 10⁵ cells/ well in 96-well flat-bottom plates. Tv was reconstituted in complete RPMI media as described above and added at the indicated MOI for the indicated period of time. After the co-culture, monocyte death was measured by determining mammalian-specific lactate dehydrogenase (LDH) release using the CytoTox-One © homogeneous membrane integrity assay (Promega) according to the manufacturer’s instructions. Samples were read on a Victor³ 1420 plate reader (Perkin-Elmer) to generate mean fluorescence intensity (MFI) values correlating with LDH presence in the supernatants. Percent death was calculated as (MFI from co-culture supernatants- MFI from live monocytes alone supernatants)/ (MFI from detergent solubilized monocytes alone supernatants–MFI from live monocytes alone supernatants) *100. Transwell cytotoxicity experiments were conducted as described above.

Human monocytes isolated by Rosette Sep © negative selection were plated at 5 x 10⁴ cells/ well in 96-well u-bottom plates. Tv was counted, and then reconstituted in complete RPMI media, and rendered dead, but intact as described above. Dead, intact Tv were then added to monocytes, or human monocyte derived macrophages (HMDM) at an MOI (multiplicity of infection) of 1, and allowed to incubate overnight (16 hours). Positive controls were 100 ng/ml LPS (sigma) 10 μg/ml poly (I:C) (Tocris) or 1000 U/ml IFN gamma (Biolegend). Subsequently plates were centrifuged and supernatants were harvested and frozen at -80°C. Supernatants were then thawed on ice and analyzed using Cytometric Bead Array (Becton-Dickenson) for IL-8, IL-6, IL-1β, TNFα, and IL-12 according to the manufacturer’s instructions. IL-6, IL-1β, and IL-12 were multiplexed, and IL-8 was measured separately on supernatants diluted 1:100. Data were analyzed using FlowJo (Treestar) to determine MFI, which was normalized to absolute concentrations according to a standard curve generated using lyophilized protein provided with the kit. IL-23 was measured using Legend Max Human IL-23 (p19/p40) ELISA kit with pre-coated wells (Biolegend) according to the manufacturer’s instructions. Wells were read using a Victor³ 1420 plate reader (Perkin-Elmer), and MFI was normalized as described above.