Bio plex pro mouse cytokine 23 plex assay

Quantification of secreted cytokines and chemokines was assessed using the Bio-Plex Pro mouse cytokine 23-plex assay (Bio-Rad). BMDM were seeded in a 24-well plate as described above. The cells were infected as described above with wild-type NMII or dotA::Tn NMII at an MOI of 10 or were treated with 2.5 ng/mL IFN-γ and 100 ng/mL LPS. Supernatants were collected at 24 h postinfection/treatment in parallel with mock-infected BMDM. Secreted cytokines and chemokines present in the cell culture supernatants were measured using the Bio-Plex Pro mouse cytokine 23-plex assay (Bio-Rad) according to the manufacturer’s instructions. Per the manufacturer’s recommendations, undiluted supernatants were measured in triplicate and quantified using a five-parameter logistic regression with an 8-sample standard curve. Results were calculated as the average of triplicate samples in pg/mL. The mean protein secretion from three independent experiments for each condition was normalized to that of the positive control (IFN-γ plus LPS). Data for secretion of MIP-1β is not reported, as it was outside the limit of detection for the positive control. Error bars represent standard deviations from the mean, with asterisks denoting P values calculated by two-way analysis of variance (ANOVA), with Dunnett’s test for multiple comparisons.

The concentration of selected inflammatory cytokines and chemokines in the serum of mice was analyzed as described previously [47 ] by a commercially available Bio-Plex Pro™ Mouse Cytokine 23-plex Assay (BIO-RAD, Irvine, CA, USA) according to the manufacturer’s instructions. The Bio-Plex Pro™ Mouse Cytokine 23-plex Assay measures the concentration of 23 cytokines and chemokines: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL12p40, IL-12p70, IL-13, IL-17A, eotaxin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), keratinocytes-derived chemokine (KC, also known as chemokine (C-X-C) motif ligand 1 (CXCL1)), monocyte chemoattractant protein (MCP)-1 (CCL2), macrophage inflammatory proteins (MIP)-1α (CCL3) and MIP-1β (CCL4), regulated on activation/normal T cell expressed and secreted (RANTES, CCL5), and tumor necrosis factor (TNF). The absorbance of the Bio-Plex Pro™ Mouse Cytokine 23-plex Assay was evaluated at Luminex BIO-PLEX 200 System (Bio-Rad, Hercules, CA, USA) [47 ]. Samples were measured as duplicates, and the mean value was used.

Quantification of secreted cytokines and chemokines was assessed using the Bio-Plex Pro mouse cytokine 23-plex assay (Bio-Rad). BMDM were seeded in a 24-well plate as described above. The cells were infected as described above with wild-type NMII or dotA::Tn NMII at an MOI of 10 or were treated with 2.5 ng/mL IFN-γ and 100 ng/mL LPS. Supernatants were collected at 24 h postinfection/treatment in parallel with mock-infected BMDM. Secreted cytokines and chemokines present in the cell culture supernatants were measured using the Bio-Plex Pro mouse cytokine 23-plex assay (Bio-Rad) according to the manufacturer’s instructions. Per the manufacturer’s recommendations, undiluted supernatants were measured in triplicate and quantified using a five-parameter logistic regression with an 8-sample standard curve. Results were calculated as the average of triplicate samples in pg/mL. The mean protein secretion from three independent experiments for each condition was normalized to that of the positive control (IFN-γ plus LPS). Data for secretion of MIP-1β is not reported, as it was outside the limit of detection for the positive control. Error bars represent standard deviations from the mean, with asterisks denoting P values calculated by two-way analysis of variance (ANOVA), with Dunnett’s test for multiple comparisons.