Elisa kits for il 6

After the rats were killed, the samples of whole blood were collected from the abdominal vein. Blood was allowed to clot for 30 min. Then, the serum was separated via centrifugation at 1,500 g for 10 min. The levels of cytokines in the serum were measured using enzyme-linked immunosorbent assay (ELISA) kits for IL-6, IL-1β, and TNF-α (R&D Systems, USA). All ELISA procedures were performed according to the manufacturers’ protocols.

Cell supernatants were obtained for the determination of IL-6 and CXCL2 protein levels. Analysis was performed using a microplate reader (PowerWave X, BioTek Instruments, Winooski, VT, USA), which measured absorbance at 450 nm, and commercially available enzyme-linked immunosorbent assay (ELISA) kits for IL-6 (R&D Systems, Abingdon, UK) and CXCL2 (Abnova, Taipei, Taiwan) according to the manufacturer’s instructions. An automated cell counter (Moelab, Hilden, Germany) was used to determine cell counts, which were used to normalize the measured protein concentrations.

Concentrations of cytokines and chemokines in cell supernatants were measured using commercially available ELISA kits for IL-6 (range of detection 9.4–600 pg/mL), TNF-α (15.6–1000 pg/mL), and CXCL8 (31.3–2000 pg/mL) (R&D Systems, MN, USA). The results obtained were normalized for the total protein content in each well, determined in cell lysates (cells lysed with 0.1% Triton X-100) by a Bradford assay, with the standard curve performed with bovine serum albumin. Cytokine release was expressed as pg or ng of cytokine/mg of total proteins.

Murine bone marrow derived macrophages (BMM) were generated as previously described [8] (link) Briefly, bone marrow cells from C57BL/6 mice were grown in DMEM/F-12 medium (Lonza) with 10% FBS (HyClone), 2 mM glutamine, 10% L-cell conditioned medium (LCM) for 7 days of differentiation at 37°C with 5% CO₂. For infection, macrophages were plated onto 24-well plates (3×10⁵ per well). Bacteria were resuspended in DMEM/F-12 medium containing 5% LCM and sonicated twice for 5 seconds each before addition to adherent monolayers. Each bacterial strain was used for infection in triplicate at an MOI = 10 and infection of macrophages was carried out for 4 hours as previously described [8] (link). To determine intracellular CFU, one set of infected macrophages was lysed in PBS containing 0.5% Triton X, and plated onto 7H10 agar plates containing the appropriate antibiotics. For stimulation of macrophages with recombinant proteins, endotoxin-free GroEL2 and GroEL2(cl) in 5% LCM were added to C57BL/6 or TLR2^-/- BMM for 24 hours. Cell-free supernatants from macrophage monolayers were isolated at various time points and assayed for cytokines by ELISA kits for IL-6, IL1-β, and TNF-α (R&D Systems, Minneapolis, MN). Assays were carried out according to manufacturer's instructions. Uninfected macrophages were used as controls for each experiment.