Mouse premixed multi analyte kit

The concentration of murine CXCL1, CXCL2, and IL-6 in mouse plasma was measured using a mouse premixed multi-analyte kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Fluorescence intensities were measured using a MAGPIX multiplexing system (Luminex Corporation, Austin, TX, USA). The concentration of murine C-reactive protein (CRP) in mouse plasma was measured by the enzyme-linked immunosorbent assay (ELISA) using CRP ELISA kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Optical densities were measured at 450 nm using a Bio-Rad Model 550 microplate reader (Bio-Rad Laboratories, Irvine, CA, USA). The concentration of analytes in plasma was calculated from standard curves generated by a curve-fitting program.

Plasma samples were analyzed using a multiplex bead analysis. C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 4 (CCL4), C-C motif chemokine ligand 5 (CCL5), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-17 (IL-17), and tumor necrosis factor α (TNF-α) were measured according to the manufacturer‘s instructions. Briefly, the plasma samples were incubated with premixed beads overnight at 4°C, and washed beads were further incurred with detection antibody for 1 h at room temperature followed by incubation with streptavidin–phycoerythrin for 30 min at room temperature. The samples were analyzed by using Luminex FLEXMAP 3D (Luminex, USA). The levels of CCL2?, CCL4?, CCL5, G-CSF, GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, and TNF-α were detected using a Mouse Premixed Multi-Analyte Kit (RD Systems Inc, USA; Cat# LXSAMSA-15).

The intratumoral CCL2, CCL5, CCL7, CXCL1, CXCL2 and VEGF were measured using Luminex liquid suspension chip. Mouse Premixed Multi-Analyte Kit (#LXSAMSM-06, R&D systems) was used in accordance with manufacturer’s instructions. Briefly, the lysate of tumor tissue was embedded with microbeads for 2 h, and then incubated with detection antibody for 1 h. Subsequently, streptavidin-PE was added into each well for 30 min, and values were read using the Bio-Plex MAGPIX System (Bio-Rad). The intratumoral CXCL9 (#MCX900, R&D systems) and CXCL5 (#ab100719, Abcam) were analyzed with commercial ELISA Kit according to the manufacturer's instructions.

The levels of serum cytokines including IL‐6 and MCP‐1 were analysed using a Mouse Premixed Multi‐Analyte Kit (R&D Systems) according to the manufacturer's instructions. In brief, 50 µL of sample or standard was added to each well of a 96‐well plate and mixed with 50 µL of microparticle cocktail. The plate was incubated at room temperature for 1 hour. After repeated washing, each sample was incubated with 50 µL of biotin‐antibody cocktail and then with 50 µL of streptavidin‐PE at room temperature for 1 hour. After washing, samples and standards were read on a Luminex 200 analyzer.

The concentrations of a set of cytokines and chemokines were determined in exudates collected from mice 1 h after i.m. injection of 20 µg of either D. russelii or B. asper venom. Exudate samples collected from five mice injected with each venom were pooled. Quantification was performed by Luminex Assays (Mouse Premixed Multi-Analyte Kit, R&D systems, Minneapolis, MN) following the methodology recommended by the manufacturer. Since no exudate develops from control mice receiving PBS injections, plasma from normal mice was used as control.