Human ccl5 rantes duoset

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.

Baseline blood specimens were collected from storage in July 2012. The sera were snap frozen and stored at −70 °C until further analysis and underwent only one freeze-thawing cycle. Baseline serum levels of CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC were determined by commercially available ELISA kits from R&D systems (Abingdon, United Kingdom; Human CCL3/MIP-1 alpha Quantikine ELISA Kit, ≤8.9 % intra-assay variation, Human CCL 5/RANTES DuoSet and Human CCL18/PARC DuoSet). CCL3 ELISA was performed according to the manufacturer’s instructions. For CCL5 measurements, 0.75 μg/ml capture antibody and 10 ng/ml detection antibody were used, for CCL18 we used 1.5 μg/ml capture antibody and 200 ng/ml detection antibody.