Magpix xponent 4.2 system

Among the total subjects whose PUFA levels were measured, a subsample of 65 patients with BD and 90 controls (see supplementary Table S1 for demographic and clinical data) underwent measurement of plasma IL-6 and TNFα. Plasma samples were obtained as described above, frozen in small aliquots, and stored at −80 °C until analysis. Investigators were blinded to the subjects’ clinical information, and IL-6 and TNFα measurement was performed using the MAGPIX CCD imaging system (MAGPIX xPONENT 4.2 system, Merck & Co., Inc.; Bio-Rad Laboratories, Inc.) using a magnetic on-bead antibody for each cytokine. Experiments were performed according to the manufacturer’s instructions.

TDCC was conducted as described above. After 24 h, the culture medium was collected. Human cytokine levels were measured using the MAGPIX ®xPONENT®4.2 system (Merck, Kenilworth, NJ, USA) with the Human Cytokine Magnetic 10-Plex Panel (Thermo Fisher Scientific) according to the manufacturer’s instructions. The data analysis was conducted using Microsoft Excel 2008 and figures were drawn using GraphPad Prism 8.4.3.

Gene expression and protein analysis was performed as described by Takahashi et al. [8 (link)]. For the quantification of mRNA, TaqMan real-time polymerase chain reaction (PCR) amplification reactions were performed using an Applied Biosystems 7500 Fast Real-Time System (Thermo Fisher Scientific Inc., Waltham, MA). All primers and the TaqMan Universal PCR Master Mix were obtained from Thermo Fisher Scientific. Specific primers for the mouse genes Gapdh (Mm99999915_g1), interleukin- (IL-) 6 (Mm00446190_m1), TNFα (Mm00443258_m1), and IL-1β (Mm00434228_m1) were used. Gene expression levels were normalized to the Gapdh expression level.
To measure the protein expression, the amounts of IL-6 and IL-1β in RAW264 cells were assayed in the supernatant of the cell culture medium using the Bio-Plex Pro™ Mouse Cytokine Assay kit (BIO-RAD, USA) with a MAGPIX xPONENT 4.2 system (Merck Millipore Co, USA).