Bio plex suspension array system

CD14⁺ macrophages were depleted from colonic MMC by positive selection (CD14 Microbeads, Miltenyi Biotech). Total MMC and CD14⁻ MMC (0.25x10⁶ cells/well) were cultured in triplicate in RPMI/10 % FCS/5 % human AB serum in the presence or absence of LPS (1 μg/mL; Sigma) for 18 h. Cytokine and chemokine levels (pg/ml) in culture supernatants were quantified using the Bio-Plex Suspension Array System according to manufacturer’s instructions (Bio-Rad).

Serum samples were collected and stored at − 80 °C freezer until analysis. Human Premixed Multi-Analyte Kit was purchased from R&D Systems (Minneapolis, Minnesota, USA). Serum CXCL13 and TGF-β were measured by Bio-Plex® suspension array system (Bio-Rad Laboratories Inc., California, USA). All samples were measured in duplicate. Four serum samples were excluded from this analysis as the volume were not enough for analysis. Two CXCL13 detection results were also excluded as they were reported with warning after bio-plex analysis. Eventually, 61 results of CXCL13 and 63 results of TGF-β were included in the following analysis.

Levels of IFN-γ and IL-10 were detected in plasma samples by a multiplex assay (Bio-Plex assay, Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions using the Luminex system (Luminex Corporation, Austin, TX, USA) and analysed with a Bio-Plex suspension array system (Bio-Rad Laboratories). Fluorescence intensity was transformed into cytokine concentration using the Bio-Plex manager software (version 3.0). A minimum of 100 beads per region were analysed. A curve fit was applied to each standard curve according to the manufacturer’s manual and sample concentrations were interpolated from the standard curves. The limit of detection was 0.79 pg/mL for IFN-γ and 2 pg/mL for IL-10.

AH samples from nAMD patients were collected before the first IVA (pre-IVA) and before the third IVA (post-IVA). At each sampling, approximately 0.1 ml of undiluted AH was collected by performing an anterior chamber limbal paracentesis. In controls, undiluted AH samples were obtained at the beginning of cataract surgery. No complication associated with sampling of AH occurred. The AH samples were stored at −80°C until processing. Twenty-seven types of inflammatory cytokines expected to provide a comprehensive coverage of inflammatory mediators (Bio-Plex Human Cytokine 27-plex panel; Bio-Rad, Hercules, CA, USA) were measured by a multiplex bead analysis system (Bio-Plex Suspension Array System; Bio-Rad) according to manufacturers’ instructions. All standards and samples were assayed in duplicate. Levels of AH cytokines below detectable levels were treated as 0 for statistical analysis (5 (link), 17 (link), 18 (link)).

Bio-Plex Suspension Array System produced by Bio-rad Laboratories Inc, based on a Microplate-ELISA bioassay system was utilized for analysis of each biomarker as previously reported [31 (link)]. The Human Diabetes assay Panel was utilized for Adipokine, leptin, ghrelin analysis [31 (link)]. Plasma-blood adiponectin, leptin, and ghrelin assays were performed with an intra-assay CV of 4%, 3%, and 4%, respectively (Bio-Rad Laboratories, Inc, Berkeley, CA, USA). Analysis were purchased from BioClarma-Research and Molecular Diagnostics , Turin.

Lysates and media were collected for multiplex protein array analysis. Lysates were collected in RIPA buffer + 0.5% bovine serum albumin. Samples were analyzed using a system that uses microbeads and flow cytometry (Bio-Plex Suspension Array System, Bio-Rad Laboratories Inc., Hercules, CA). Fluorescent-coded beads are conjugated to defined antibodies that recognize the cytokines/chemokines in this quantitative technique. The cell lysates or control media were first incubated for 90 min with all of the microbeads types. After washing, the samples were incubated with biotinylated secondary antibodies also specific for the target cytokines for 30 min. The samples were washed again, incubated with streptavidin-coupled phycoerythrin reporter for 10 min, and then subjected to a final wash. The samples were then diluted in buffer and underwent flow cytometry analysis. At least 100 microbeads were assayed for every sample. The concentrations of each cytokine were calculated based on the inclusion of a standard curve with defined amounts of every analyte.