CellTiter-Glo™ luminescent assay (Promega, Madison, WI, USA) was performed according to manufacturer's instruction to quantify ATP levels of metabolically active adherent cells.
A commercially available enzyme-linked immunosorbent assay (ELISA) kit (DRG Instruments, Marburg, Germany) was used manufacturer instruction to quantify the amount of estrone EIA-41744) in supernatants from stimulated C-28/I2 cells.
Each participant received by mail a short photograph atlas and guidance notes to estimate portion sizes (with instructions to quantify the portions used by children) developed on the basis of EPIC-SOFT picture book32 and a hard-copy diary structured by meal, where all the information on food consumption the day before the telephone interview was self-recorded.
An ABI SYBR® Green master mix in conjunction with an ABI StepOne Plus machine was used according to the manufacturer's instructions to quantify Glo1 and beta-actin cDNA expression levels.
PicoGreen dscDNA Quantitation kit (Molecular Probes, Eugene, OR, USA) was used according to the manufacturer's instructions to quantify and standardize PCR product concentrations.
Enzyme-linked immunosorbent assays (ELISA) were used according to manufacturer's instructions to quantify IL-6, sCD14 (R&D Quantikine®, HS600B and DC140 respectively) and hs-CRP (Calbiotech®, CR120C).
After incubation for 24 h at 37°C in 5% CO2 and 95% humidified air, the apoptosis assay was then carried out according to the manufacturer's instructions to quantify cytoplasmic histone-associated DNA fragments.
The LDH-release assay (CytoTox 96 Non-Radioactive Cytotoxicity Assay; Promega Corporation, Madison, WI, USA) was used per the manufacturer's instructions to quantify cytolysis and to validate cell-viability measurements obtained with MTT assay.
Mouse VEGF-A (BMS619; eBioscience, San Diego, CA), mouse IL-12 (KMC0121; Invitrogen), mouse IFN-γ (KMC4021; Invitrogen), and mouse placental growth factor (E0114m; Uscn Life, Wuhan, China) ELISAs were performed according to the manufacturer's instructions to quantify VEGF, IL-12 and IFN-γ, and PlGF, respectively.
The Griess reaction (Griess Reagent System, Promega; Madison, WI) was used according to manufacturer's instructions to quantify the nitrite concentration in the supernatant of thioglycolate-elicited peritoneal macrophages (plated at a density of 200,000 cells per well in 96-well plates) co-incubated with mycobacterial (MOI = 10 1).
The manufacturer's instructions were followed to quantify the relative gene expression.
