Human il 1β il 1f2 duoset

An enzyme-linked immunosorbent assay was performed to detect and quantify inflammatory cytokines in supernatants from cocultures of ovarian cancer cell lines and PBMC. ELISA kits were purchased from R&D Systems (Human IL-1β/IL-1F2 DuoSet, Human TGF-β DuoSet, Human TNF-α DuoSet, Human IL-12 p70 DuoSet ELISA) and were used according to the manufacturer’s protocol. The optical density of the colorimetric reaction was determined using an ELISA plate reader at 450 nm. Recombinant human IL-1β, IL-12, TNF-α, and TGF-β were used to establish the standard curve for each assay.

Cytokines were preliminarily measured in cell culture supernatants (400 μL) of two samples, control and treated with the oxysterols mixture, 20 μM in ethanol, by a semi‐quantitative method to simultaneously detect the relative levels of 36 different cytokines and chemokines using the Human Cytokine Array Panel A (R&D systems, Abingdon, UK) following the manufacturer instructions. The membranes were developed using ImageQuant LAS 4000 mini. Signal intensities of each membrane array were analyzed using the ImageQuant software (Molecular Dynamics, Amersham Pharmacia Biotech). Three cytokines were selected for quantitative analysis by ELISA on the basis of the initial screening process described earlier. Levels of human cytokines IL‐1β, MIF, and RANTES were quantified using appropriate kits (“Human IL‐1β/IL‐1F2 DuoSet,” “Human MIF DuoSet,” and “Human CCL5/RANTES DuoSet” R&D systems, Abingdon, UK), following the manufacturer's instructions.

The amount of IL-1β, IL-6, VEGF and BDNF secreted by astrocytes after the treatment with MS and healthy CSF were determined to confirm our qPCR results. Conditioned media of the experiment mentioned above was used for this purpose. Human IL-1β/IL-1F2 DuoSet, Human VEGF DuoSet, Human Total BDNF Quantikine ELISA kits (R&D Systems, USA) and Human Interleukin-6 ELISA Kit (SUNLONGBIOTECH, China) were used for this purpose, following their manufacturer’s protocol. Optical density was measured at 450 nm with correction at 570 nm. Concentrations were calculated using standard curve equations for each set and then analyzed using GraphPad Prism statistical analysis software.