Cytometric bead array cba mouse inflammation kit

The chemokines MCP-1, MIG, IP-10, IL8 and complement system anaphylatoxins C3a, C4a and C5a were measured in human serum from patients with severe, moderate and mild form of acute pancreatitis. Measurements were performed with CBA Kits from BD (Human Anaphylatoxin Kit 561418BD; Human Chemokine Kit 552990; BD San Jose, USA). Murine chemokines and cytokines were measured by BD cytometric bead array (CBA) mouse inflammation kit in serum of mice.

After injection of EB dye to analyze BBB integrity (described above), blood was collected through the orbital plexus, after local application of anesthesia eye drops. The collected blood was centrifuged to obtain serum, divided into aliquots, and stored in a freezer at -80°C until use. To measure serum cytokine levels, the BD Cytometric Bead Array (CBA) Mouse Inflammation kit (catalogue 552364, BD Bioscience) was used, according to the manufacturer’s recommendations. The kit was used for the simultaneous detection of interleukin 6 (IL-6), interleukin 10 (IL-10), interferon gamma (IFNγ), tumor necrosis factor (TNF), interleukin 12 (IL-12) and monocyte chemotactic protein (MCP-1/CCL2), in a single sample. The cytokine standards were diluted serially to construct the calibration curves, used to determine the concentrations of the cytokines. The samples were analyzed using the 13-Color CytoFLEX-S flow cytometer (Beckman-Coulter, USA). Individual cytokine concentrations were indicated by their fluorescent intensities and expressed in pg/mL, using the FCAP Array Software. The theoretical limits of detection were: 5 pg/mL for IL-6, 2.5 pg/mL for IFNγ, 7.3 pg/mL for TNF, 10.7 pg/mL for IL-12 and 17.5 pg/mL for IL-10.

Triglycerides, total cholesterol, and glucose were analyzed in the plasma samples. The analysis was carried out by enzymatic methods using commercial kits (Wiener Lab, Rosario, Argentina). Also, plasmatic leptin levels were determined by enzyme-linked immunosorbent assay (Mouse / Rat Leptin Quantikine ELISA Kit, MN, USA). Plasma insulin levels were measured using ELISA (Mouse Insulin ELISA Kit, Alpco Diagnostics, USA). The chemokine monocyte chemoattractant protein- 1 (MCP-1) and the cytokines, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6 (IL-6), and IL-10 were determined by flow cytometry using the BD™ Cytometric Bead Array (CBA) Mouse Inflammation Kit (BD Bioscience, 560485, San Diego, CA, USA).

Serum cytokine levels were analyzed using BD cytometric bead array (CBA) mouse inflammation kit (BD Pharmingen, San Diego, CA, USA) on a FACS Calibur Cytometer and analyzed using BD FCAP Array software (BD Bioscience). The cytokine kit covered mouse IL-6, IL-10, IL-12p70, TNF, IFNγ, and MCP-1.

The levels of IFN-γ, TNF-α, interlukin-6 (IL-6), IL-10, IL-12p70, and monocyte chemoattractant protein-1 (MCP-1) in bronchoalveolar lavage (BAL) fluid or lung tissue were analyzed using a BD Cytometric Bead Array (CBA) Mouse Inflammation Kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions with the exception that a total of 2 μl of each capture bead was used in 50 μl of BAL sample and the PE-detection reagent was diluted 1 in 5. Samples were analyzed using a Becton Dickinson FACSCalibur flow cytometer and data analyzed using the FlowJo software package (Tree Star, Inc., Ashland, OR, USA).

For measurement of intracellular cytokines, dendritic cells and CD4⁺ T cells were co-cultured and stimulated for 3 days with heat-killed H37Rv at 0.1 MOI or overnight with anti-CD3 and anti-CD28 antibody in culture media. H37Rv strain was generously provided by Sang-Nae Cho (Yonsei University). Brefeldin A Solution (1000 x) Thermo Fisher Scientific, Waltham, MA, USA) was added for 4 h before harvest and then harvested cells were stained with PerCP-Cy™5.5 rat anti-mouse CD4 (BD biosciences, California, USA) or PE rat anti-mouse CD8α (BD biosciences, California, USA). Stained cells were permeabilized with IC Fixation Buffer (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's recommendations. Next, permeabilized cells were stained with TNF-α mAb, APC (Thermo Fisher Scientific, Waltham, MA, USA) and IFN-γ mAb (XMG1.2) PE (Thermo Fisher Scientific, Waltham, MA, USA).
The supernatants of homogenized tissues were analyzed for cytokine production using BD™ Cytometric Bead Array (CBA) Mouse Inflammation kit (BD biosciences, California, USA) according to the manufacturer's instructions.

CD4⁺ T cells were isolated by MACS from the spleens of Pbx1^ΔT and Pbx1^fl/fl mice, as described above, and cultured for 72 hours with no stimulation or under Th1 or Tr1 cell polarizing conditions, as previously described (82 (link)). Cell culture supernatants were then collected for measurement of IFN-γ and IL-10 levels. Cytokine levels were assessed using the BD Cytometric Bead Array (CBA) Mouse Inflammation Kit or Mouse Th1/Th2/Th17 Cytokine Kit (BD Biosciences) as per manufacturer instructions. Serum samples from mouse blood were used undiluted, while cell culture supernatants were diluted 1:5 in 1× PBS for the detection of most cytokines. Supernatants were diluted 1:50 in 1× PBS for the detection of IFN-γ. CBA data were analyzed using the FCAP Array Software v3.0 (BD Biosciences). Human IFN-γ in whole-blood assays was measured by ELISA, as previously described (54 (link)).