Multiplex immunoassay

To measure serum ENL and END concentrations, samples underwent solid-phase extraction and overnight enzymatic hydrolysis. The unconjugated lignans were then isolated by solid-phase extraction, converted to tert-butyldimethylsilyl ethers, and analyzed by gas chromatography mass spectrometry [28 (link)]. Serum cytokines were analyzed by Bio-Plex Multiplex Immunoassay on a Bio-Plex^Ⓡ Magpix Multiplex Reader (Bio-Rad, Inc., Hercules, CA, USA).

To evaluate adaptive immune function, CD4+ T cells were isolated and stimulated as follows. Peripheral blood mononuclear cells were collected with density gradient centrifugation by using lymphocyte-separation medium (Mediatech, Manassas, VA, USA). CD4⁺ T cells were then isolated with negative selection by using magnetic beads (Miltenyi Biotec, Auburn, CA, USA). T cells, 5 × 10⁵ CD4⁺, were incubated for 24 hours at 37°C in complete media (RPMI + 10% fetal bovine serum + 1% penicillin/streptomycin) in the presence or absence of 10 μg/ml of phytohemagglutinin (PHA) (Sigma, St. Louis, MO, USA). After 24 hours, the supernatant was collected and stored at −80°C for subsequent cytokine analysis. Production of the cytokines IL-2, IL-4, IL-10, and IFN-γ from stimulated cells was determined with multiplex immunoassay (Bio-Rad, Hercules, CA, USA).
Percentage of regulatory T cells (CD4⁺, CD25⁺, CD127^lo) among freshly isolated, unstimulated CD4⁺ T cells was determined with flow cytometry. The following antibodies were used: FITC anti-human CD4, APC anti-human CD25, and PE anti-human CD127 (Becton Dickinson). Data were acquired on an LSRII cytometer (Becton Dickinson) and analyzed by using FlowJo software (TreeStar, Ashland, OR, USA).