Overnight cultures of SL1344 mcGAS and SL1344mcGASAA were continued by taking one optical density (OD) and added to 10 mL MgM-MES pH7.4 (170 mM MES (2-(N-morpholino) ethanesulfonic acid), 5 mM KCl, and 7.5 mM (NH4)2SO4, 0.5 mM K2SO4, 1 mM KH2PO4, 8 µM MgCl2(6xH2O), 38 mM glycerol, and 0.1% casamino acids) supplemented with antibiotics until cultures reached again ~OD 1. Bacteria were pelleted at 10 min, 5000 rpm, and supernatant was taken for cGAMP measurement according to 2′-3′-cGAMP ELISA Kit manufacturer (Cayman chemical). THP-I and DC supernatants were harvested 18–24 hours after stimulation, and secretion of IFN-β was measured by a human IFN-beta DuoSet ELISA (R&D Systems) according to the manufacturer’s instructions.
Human ifn beta duoset elisa
Manufactured by R&D Systems
The Human IFN-beta DuoSet ELISA is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed to measure human interferon-beta levels in cell culture supernatants, serum, and plasma.
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3 protocols using human ifn beta duoset elisa
1
Quantification of cGAMP and IFN-β
2
Quantitative IFN-β Immunoassay Protocol
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Growth medium supernatants were centrifuged to exclude floating cells. Resulting supernatants were 20X up-concentrated by centrifuge filtering through 10 kDa cut-off columns (Amicon Ultracel-10, Merck). ELISA (Human IFN-beta DuoSET ELISA, R&D Systems) was conducted according to supplier’s protocol. Optical density was measured at 450 nm with pathlength correction at 540 nm in a microplate spectrophotometer (PowerWave XS2, BioTek) coupled to the Gen5 software v2.09.1. IFN-β standards were included in all experiments, and a best-fitting 2nd degree polynomial function was used for calculation of measured IFN-β in the samples.
3
C. parvum Infection Modulates Interferon Responses
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96 well HCT-8 cultures grown to 60% confluency were infected with 25,000 C. parvum oocysts. At the indicated timepoint post infection, supernatants were removed and spun at 1,000xg for 10 minutes to pellet debris. Supernatants were frozen at -80°C. IFNβ and IFNλ protein levels from HCT-8 cultures were measured by Human IFN-beta DuoSet ELISA (DY814, R&D Systems) and Human IL29/IL28B (IFN-lambda 1/3) DuoSet ELISA (DY1598B, R&D Systems). Protein levels of IFNλ from intestinal biopsies were measured by Mouse IL28B/IFN-lambda 3 DuoSet ELISA (DY1789B, R&D Systems). Protein levels of IL-18 from intestinal biopsies were measured by ELISA (BMS618-3, ThermoFisher, Waltham, MA). Assays were performed according to the manufacturer’s instructions.
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