Luminex technology

IL-1β, IL-6, IL-10, keratinocyte chemoattractant (KC), tumor necrosis factor (TNF)-α, and macrophage inflammatory protein (MIP)-2 levels were determined in mouse plasma and brain homogenates with Luminex® technology (Bio-Rad Laboratories). Expression of MASP-2 and C5b-9 was measured in mouse brain homogenates by ELISA (CUSABIO and USCN Life Science, respectively). Albumin concentrations in brain homogenates were determined with ELISA (ALPCO Diagnostics).

CXCL10 was measured in tissue and plasma using Luminex technology (Bio-Rad, Hercules, CA, USA). The tissue level of CXCL10 was expressed as pg/mg of protein. The plasma CXCL10 levels of CRC patients and controls were expressed as pg/ml.

For the analysis of DAMP and cytokine release, MCA205 cells were seeded at 3 × 10⁵ cells/well in 6-well tissue culture plates in 2 ml of medium. Supernatant from dying cells was collected at indicated time points and stored at −20 °C. ATP release was measured using CellTiter-Glo^® Luminescent Cell Viability Assay (#G7570, Promega, USA). LDH release was measured using colorimetric Pierce LDH cytotoxicity assay (#88954, Life Technologies, USA) according to the manufacturer’s protocol. Both ATP and LDH results were normalized to values obtained from untreated (live) cells. HMGB1 release was performed using ELISA assay (#ST51011, Tecan, Switzerland) according to the manufacturer’s instructions. Mouse cytokines in cell culture supernatants were determined by a magnetic bead-based multiplex assay using Luminex technology (Bio-Rad, Hercules, CA, USA).

Tissue samples were weighed and were homogenized in 500 μl PBS with protease inhibitors, after which lysis was completed by addition of lysis buffer (20 mM Tris HCl (pH 7.4), 200 mM NaCl, 1% Nonidet P-40) and incubation for 20 min on ice. Full-speed centrifugation for 30 minutes cleared the homogenate and supernatant was used for further analysis. Mouse cytokines in cell culture supernatants and tissue homogenates were determined by magnetic bead-based multiplex assay using Luminex technology (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Cytokines from tissue homogenates were normalized to weight of tissue, while cytokines from cell culture supernatants were expressed as concentration per ml of cell culture medium.

Human IL-1β and IL-18 cytokine levels were determined in cell culture supernatants by magnetic bead-based multiplex assay using Luminex technology (Bio-Rad). The IL-1β and IL-18 ratios were calculated by dividing the cytokine level of the combined colchicine TcdA treatment by the cytokine level of the treatment with TcdA alone. GraphPad Prism V.6.0 software was used for data analysis.

For the study we used a bead-based assay based on Luminex technology (Bio-Rad, Milan, Italy), that allowed to measure both CXCL10 and IL-6 using a low volume of serum (50 µl). To minimize inter-assay variation, donor sera was assayed at the end of the study using the same commercial lot. Appropriate pool of sera was used to estimate intra-assay and inter-assay coefficient of variation.