Bio plex 200 reader

The bead based multiplex suspension assay was performed as described by Khurana et al., [22 ]. Briefly 18 different scFv antibody-coupled beads were mixed and dispensed in pre-wet wells of 96-well filter bottom plates (Multiscreen BV; Millipore, Billerica, MA) containing serially diluted termite extract. Antibody-coupled beads were incubated with termite extract for 1 hr in the dark at room temperature with gentle mixing. The binding of scFv and proteins was detected with a mixture of rabbit antisera containing anti-E6Cg, anti-Bla g 1, anti-Bla g 2, and anti-Bla g 4 (each diluted 1:500) [23 (link)]. Biotin-coupled anti-rabbit IgG antibody (KPL, Gaithersburg, MD) was diluted (1:1000) and added to each well for detection. The binding was detected using diluted streptavidin-R-Phycoerythrin (100 μg/mL) (Thermo Scientific). Finally, the beads were resuspended in sheath fluid (Bio-Rad Laboratories) and median fluorescence intensities (MFI) were measured in a BioPlex 200 reader (Bio-Rad Laboratories).

The mitochondrial Oxidative Phosphorylation (OXPHOS) pathway was evaluated using the MILLIPLEX^® MAP Human OXPHOS Magnetic Bead Panel (Merck, Darmstadt, Germany). This kit, based on the Luminex xMAP^® technology, permits the simultaneous detection of NADH-ubiquinone oxidoreductase (Complex I), succinate ubiquinone oxidoreductase (Complex II), ubiquinone cytochrome c oxidoreductase (Complex III), cytochrome c oxidase (Complex IV), ATP synthase (Complex V) and nicotinamide nucleotide transhydrogenase (NNT).
Samples were analyzed with the Bio-Plex^® 200 reader and data were computed using Bio-Plex Manager^® software (BIO-RAD Laboratories, Hercules, CA, USA), which express data as MFI. The samples preparation and quantification were performed following the manufacturer’s instructions.

The MILLIPLEX^® MAP 6-plex magnetic bead signaling kit (EMD Millipore Corporation, Billerica, MA, USA), was used to detect changes in phosphorylated NF-κB (Ser536), FADD (Ser194), IKKα/β (Ser177/Ser181), IκBα (Ser32) and in total protein levels of TNFR1 and c-Myc, in HT29 cell lysates. Briefly, cells were seeded in a 12-well plate at a cell density of 3.3 × 10⁵ per well, pre-incubated or not with curcumin, and then stimulated with IFNγ. Forty-eight hours after stimulation cell were harvested, washed with phosphate-buffered saline and lysed in the provided lysis buffer supplemented with protease inhibitors. To remove particulate matter, lysates were centrifuged at 14,000× g for 20 min at 4 °C. Cell lysates were analyzed following manufacturer’s instructions using the Luminex technology. Samples were acquired with the Bio-Plex^® 200 reader (BioRad, Hemel Hempstead, UK) and data were analyzed by Bio-Plex Manager^® software, which returned data as median fluorescence intensities (MFI). Results were represented as means and standard deviations of four replicate wells.

Conditioned media were harvested and processed for cytokine analysis in duplicate with a custom Bio-Rad Bio-Plex human cytokine reagent kit for IL-1 receptor antagonist (IL-1ra), IL-6, IL-8, IL-10, IL-12, granulocyte colony stimulating factor (G-CSF), GM-CSF, interferon-inducible protein (IP-10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein 1-α (MIP-1α or CCL3), MIP-1β (CCL4), regulated and normal T cell expressed and secreted (RANTES), TNF-α, and vascular endothelial growth factor (VEGF) according to the manufacturer's instructions (Bio-Rad, Hercules, CA). Data were acquired on the Bio-Rad Bio-Plex 200 reader equipped with a magnetic workstation and analyzed using Bio-Plex software version 6.0 (Bio-Rad). Values presenting a coefficient of variation beyond 10% were discarded before the final data analysis. Minimum levels of detection (pg/mL) were 4.89 for IL-1ra, 0.23 for IL-6, 0.58 for IL-8, 0.17 for IL-10, 0.26 for IL-12, 0.1 for G-CSF, 2.26 for GM-CSF, 1.83 for IP-10, 3.56 for MCP-1, 2.38 for MIP-1α, 2.69 for MIP-1β, 0.49 for RANTES, 8.84 for TNF-α, 3.54 for VEGF.

Cytokines release was evaluated on culture supernatants samples using a magnetic bead-based multiplex immunoassays (Bio-Plex®) (BIO-RAD Laboratories, Milano, Italy) following manufacturer's instructions. The analysis included 27 cytokines and chemokines: IL-1β, Il-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17A, Eotaxin, basic-FGF, G-CSF, GM-CSF, IP-10, IFN-γ, MCP-1, MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α, VEGF. Data were acquired using the Bio-Plex 200 reader, while a digital processor managed data output and Bio-Plex Manager® software presented data as Median Fluorescence Intensity (MFI) and concentration (ng/ml) as well (BIO-RAD Laboratories, Milano, Italy).

The concentrations of TNF-α (analyzed by means of bead and antibody set with cat. 171BA015M, Bio-Rad), IL-10 (171BA004M, Bio-Rad), IL-17 (171BA005M, Bio-Rad), IFN-γ (171BA013M, Bio-Rad), and IL-22 (171BA008M, Bio-Rad) in undiluted culture supernatants were analyzed by Luminex assays (reagent kit and standards; cat. 171304090M and 171DA0001, Bio-Rad) using a Bio-Plex 200 reader (Bio-Rad) according to the manufacturer’s protocol. The upper and lower limits of quantification (ULOQ and LLOQ, respectively) for each assay were: ULOQ/LLOQ (TNF-α): 4.68 and 0.3 pg/ml; ULOQ/LLOQ (IL-10): 12.92 and 3.2 pg/ml; ULOQ/LLOQ (IL-17): 25.92 and 3.1 pg/ml; ULOQ/LLOQ (IFN-γ): 11.38 and 0.7 pg/ml; and ULOQ/LLOQ (IL-22): 41.57 and 2.5 pg/ml.