MoDC were obtained from CD14+ human monocytes purified from blood of healthy subjects as previously described.30 (link) MoDCs were seeded into round-bottom, 96-well culture plates at a density of 105 cells/well in complete RPMI medium without penicillin/streptomycin and infected with 107 CFU/mL of AIEC-LF82 or E. coli Nissle 1917 strain alone [MOI 10:1 bacteria per phagocytic cell]. Following 1 h of phagocytosis, gentamicin [20 ug/mL, Sigma-Aldrich, Darmstadt, Germany] was added to kill extracellular bacteria. Subsequently, infected MoDC were washed and maintained in RPMI supplemented with gentamicin [2 ug/mL] for additional 22 h. The levels of IL-1β [BioLegend], IL-12p70 [BioLegend], IL-23p19 [R&D Systems], IL-6 [ImmunoTools], IP-10 [R&D Systems], and RANTES [R&D Systems] in supernatants of infected MoDC were analysed after 24 h by ELISA. The ELISA plates were read on a microplate reader [SAFAS MP96], and data were analysed with Prism software [version 7; GraphPad Software, La Jolla, CA].
Protocol detail
Cytokine Production by AIEC-infected MoDCs
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Corresponding Organization : Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico
Other organizations : European Institute of Oncology, University of Milano-Bicocca, FIRC Institute of Molecular Oncology, Sapienza University of Rome
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Variable analysis
independent variables
- Infection of MoDC with AIEC-LF82 or E. coli Nissle 1917 strain (MOI 10:1 bacteria per phagocytic cell)
- Levels of IL-1β, IL-12p70, IL-23p19, IL-6, IP-10, and RANTES in supernatants of infected MoDC after 24 h
- MoDC were obtained from CD14+ human monocytes purified from blood of healthy subjects
- MoDC were seeded into round-bottom, 96-well culture plates at a density of 10^5 cells/well in complete RPMI medium without penicillin/streptomycin
- Following 1 h of phagocytosis, gentamicin [20 ug/mL] was added to kill extracellular bacteria
- Infected MoDC were washed and maintained in RPMI supplemented with gentamicin [2 ug/mL] for additional 22 h
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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