Bio plex multiplex immunoassay system

The concentrations of 30 cytokines in AH were analysed using a Bio-Plex multiple assay (Bio-Plex Multiplex Immunoassay System; Bio-Rad, Hercules, CA). Measured cytokines were bFGF, exotoxin, granulocyte colony-stimulating factor, GM-CSF, interferon-γ, IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IP-10, MCP-1, MIP-1α, MIP-1β, platelet-derived growth factor BB; regulated upon activation normal T-cell expressed and secreted tumour necrosis factor-α, VEGF-A, VEGF-C, VEGF-D and PlGF. The analysis was performed according to the manufacturer’s instructions. Standard curves were generated using the Bio-Plex TM 200 System (software version 6.1; Bio-Rad Laboratories).

The plasma lipid profile was determined by measuring the content of triglycerides (TGs), total cholesterol, high-density-lipoprotein (HDL) and low density-lipoprotein (LDL) by standard enzymatic procedures using reagent kits (Hospitex Diagnostics, Florence, Italy). Plasma insulin, ghrelin, glucose-dependent insulinotropic polypeptide (GIP), glucagon like peptide (GLP-1), plasminogen activator inhibitor (PAI)-1, IL-1β, TNF-α, IFN-γ, IL-6, IL-10 and IL-17 levels were measured by using Bio-Plex Multiplex Immunoassay System (Bio-Rad Laboratories, Hercules, CA, USA). Intestinal alkaline phosphatase (IAP) activity was detected in plasma with SensoLite pNPP Alkaline Phosphatase colorimetric assay kit (AnaSpec Inc, Fremont, CA, USA) following manufacturer’s instructions for kinetic reading.

Cytokines levels were measured in plasma by using Bio-Plexmultiplex immunoassay system (Bio-Rad,Hercules, CA), as described by us previously^{45 (link)}. RNA was isolated from epididymal fat pad using the E.Z.N.A.® Total RNA Kit (Omega Bio-Tek, Norcross, GA). The purity and concentration of the RNA were confirmed spectrophotometrically with Nanodrop (Thermo Scientific,Waltham, MA). Total RNA was converted to cDNA using the miScript cDNA synthesis kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. SsoAdvanced™ Universal SYBR® Green Supermix kit (Bio-Rad,Hercules, CA) was used to analyze gene expression, and GAPDH was used as the housekeeping gene. List of all the primers has been provided in Supplementary Table 5. Complete Blood Cell count (CBC) was performed using hematological analyzer VetScan HM5 (ABAXIS, Union City, CA). Circulating LPS level was quantified as previously described^{46 (link)}. Colonic myeloperoxidase was assessed according to the manufacturer’s instruction (Abcam, Cambridge, MA)^{47 (link)}. Free fatty acid was quantified in serum according to the manufacturer’s instruction (Zen-Bio Inc, Research Triangle Park, NC).

Plasma levels of insulin, leptin and glucagon hormones, and inflammation-related cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), were measured using a Bio-Plex Multiplex Immunoassay System (Bio-Rad Laboratories, Hercules, CA, USA), following manufacturer’s instructions.

Blood samples were collected from patients who had fasted overnight prior to starting dialysis, and plasma was separated and stored at −80 °C until analysis. Plasma levels of IL-6, TNF-α, IL-8, and MCP-1 were measured using the Bio-Plex Multiplex Immunoassay System (Bio-Rad) based on a previously published protocol [26 (link)]. Human sTLR4 levels were detected using an enzyme-linked immunoassay kit from Elabscience (Wuhan, China) following the manufacturer’s instructions. Each sample was tested in duplicate to verify results. Serum levels of albumin, calcium, phosphate, total cholesterol, urea, and creatinine were determined with a Hitachi 7600 autoanalyzer (Hitachi, Tokyo, Japan). Adequacy of dialysis was estimated by single-pool Kt/V for urea (spKt/V_urea) using the Daugirdas equation [27 (link)]. Pre-dialysis blood pressure was measured from the nonaccess arm after the patient had rested 5 min in a seated position before needle insertion for hemodialysis. The mean arterial pressure was calculated by adding one-third of the pulse pressure to diastolic pressure.

The non-transfected SH-SY5Y and the CTX-TN2A cells were plated on 96-well plates and treated with 10 µM of AmyP53 for 24 h at 37 °C. After 24 h of incubation, the supernatants were collected and chemokines and proinflammatory factors were quantified by Bioplex Multiplex Immunoassay System (Bio-Rad, Hercules, CA, USA) according to manufacturer’s instructions.