Bio plex data pro software

Levels of plasma proinflammatory cytokines IL-6, IL-12p70, TNF-α, and IFN-γ and chemokines MCP-1 and IP-10 were determined using an NHP customized multiplex system (R&D Systems, Minneapolis, USA; catalog number FCSTM21) following the manufacturer’s instructions. Briefly, the assay uses magnetic microparticles precoated with analyte-specific antibodies, which are embedded with fluorophores at set ratios for each unique microparticle region. Microparticles, standards, and plasma samples are pipetted into wells and the immobilized antibodies bind the analytes of interest. Plasma samples require a 2-fold dilution with a calibrator diluent provided in the kit. After any unbound substances are washed away, a biotinylated antibody cocktail specific to the analytes of interest is added to each well. Following a wash to remove any unbound biotinylated antibody, a streptavidin-phycoerythrin conjugate (streptavidin-PE), which binds to the biotinylated antibody, is added to each well. After a final washing step to remove unbound streptavidin-PE, the microparticles are resuspended in buffer and read using a Magpix instrument (Luminex, USA) and analyzed with the Bio-Plex Data Pro software (Bio-Rad, Hercules, CA).

In brief, 48 hrs after transfection of indicated siRNAs or microRNA mimics, EGM2 was removed and replaced with EBM2 for 3 hrs to starve the HUVECs. Subsequently, 25 ng/ml recombinant human VEGF165 (293-E-010; R&D) was added and cells were harvested at indicated time points and lysed with EDTA-free lysis buffer (Cat. 04719964001; Roche Applied Science) with 1× protease/phosphatase inhibitor cocktail (#5872; Cell Signaling). Protein concentrations were measured with the BCA protein assay kit (23227; Thermo Scientific) and diluted into 200 μg/ml. Phospho-ERK1/2 Bio-PlexPro™ assay was performed according to the manufacture’s instruction. Of each sample, 100 μl was incubated with capture antibodies (171-v50006M, 171-v60003M; Bio-Rad), and after washing, streptavidin-PE was applied for visualization. Samples were processed with the Bio-plex 200 (Bio-Rad) and data were analysed with Bio-Plex Data Pro software (Bio-Rad) and Graphpad Prism 6.0.

A commercial human multiplex luminex kit (R&D Systems) was performed according to the manufacturer’s instructions. Briefly, 50 μL of sample (diluted 1:2) and standard were added in duplicates to the relevant wells and incubated with pre-mixed microbeads for 2 h on an orbital plate shaker at room temperature. The plates were washed three times with wash buffer and 50 μL of biotinylated detection antibody added per well. Samples were incubated for 1 h at room temperature on the plate shaker. Streptavidin–phycoerythrin (PE) solution (50 μL/well) was added, and plates incubated for a further 30 min at room temperature on a plate shaker, protected from direct light. Microparticles were resuspended by adding 100 µL of wash buffer. The assay was analyzed with a Bio-Plex 200 system (Bio-Rad, Hercules, CA, USA). The results were analyzed using Bio-Plex Data Pro software (Bio-Rad).