Mouse il 6 duoset elisa

ELISA was conducted for the measurement of IL-6 and TNF-alpha levels in the serum. After euthanasia, blood was collected in BD Vacutainer™ SST tubes (Thermo, MA, USA), incubated at room temperature for 10 min, and centrifuged for 10 min at 4000 rpm at 4 °C. Separated serum samples were used for measuring the IL-6 and TNF-alpha levels. The ELISA kits used were Mouse IL-6 DuoSet ELISA (DY406–05, R&D systems, MN, USA) and Mouse TNF-alpha DuoSet ELISA (DY410–05, R&D systems, MN, USA). All experiments were conducted according to the manufacturer’s instructions.

ELISA was performed to determine the levels of IL-6 and TNF-α in the cell supernatant collected from LRRK2 parental and KO RAW 264.7 cells seeded in a 96-well plate (20,000 cell/well) after LPS treatment for 6 and 24 h. Cell supernatants were collected and stored at −80 °C. IL-6 was detected using Mouse IL-6 DuoSet ELISA (R&D systems, DY406-05) and TNF-α was detected using Mouse TNF-α DuoSet ELISA (R&D systems, DY410-05) according to the manufacturer’s protocols. The samples were diluted 30 times before use. Absorbance was measured at 450 nm and 570 nm for background correction using a Synergy™H1 Hybrid Multi-Mode Reader (BioTek^® Instruments GmbH, Bad Friedrichshall, Germany). Inflammatory cytokine levels were determined using the standard samples provided in the kit.

The ELISA kit was used to determine the concentration of cytokines in bronchoalveolar lavage fluid and plasma samples from mice. ELISA kits used were mouse IL-6 DuoSet ELISA (R&D Systems, Minneapolis, MN, USA, DY406-05) and mouse CXCL1/KC DuoSet ELISA (R&D Systems, DY453-05), and the assays were carried out as per the kit instructions. All BAL samples were run neat. Two technical replicates of all samples and standards were used. Plates were analyzed on a Thermo Scientific Varioskan^® Flash microplate reader at 450 nm, with wavelength correction at 540 nm. Cytokine concentration was calculated using interpolation of a four-parameter logistic sigmoidal standard curve, as suggested by the kit instructions.

Human Ang-2 DuoSet ELISA kit was purchased from R&D Systems, Bio-Techne. Human HS ELISA kit was purchased from AMS Biotechnology. Mouse IL-6 DuoSet ELISA and Ang-2 quantikine ELISA were purchased from R&D Systems, Bio-Techne. Mouse HS ELISA was purchased from LifeSpan Biosciences. All human and murine ELISAs were performed after a single freeze-thaw.
For experiments using statically cultured endothelial cells, absolute Ang-2 levels from supernatant are reported. However, as the flow system uses a substantially greater volume of culture medium, absolute measures of Ang-2 are impossible to interpret against experiments using static conditions. For experiments using flow-conditioned endothelial cells, Ang-2 levels were measured in supernatant sampled immediately following 48 hours of flow conditioning (baseline) and 24 hours after the reported experimental condition (24-hour sample). Absolute differences between baseline and 24-hour levels were then calculated and reported as relative values to the mean of the comparative control level.

Murine fibroblasts 3T3 L1 cells line, grown in DMEM medium (Lonza, Switzerland) supplemented with 10% heat-inactivated fetal calf serum (FCS), 50 U/ml penicillin and 50 U/ml streptomycin (Lonza, Levallois-Perret, France) were cultivated at 1 × 10⁵ cells per well during 24 h at 37°C, 5% CO_2. Then, medium was changed and bacterial preparations added at 10% for supernatants, pellet and control medium or MOI 100 for bacteria suspensions during 24 h. Supernatants of co-incubations were collected and stored at -80°C before ELISA analyses (mouse IL-6 DuoSet ELISA, R&D).

J774A.1 cells or BMDMs cultured in 48-well plates were infected with SE strains as described above, the supernatants were harvested at 4.5 h after infection. Cytotoxicity was quantified using the LDH Cytotoxicity Assay Kit (Beyotime Biotechnology Co. Ltd., Haimen, China) according to the manufacturer’s instructions. Expression of the cytokines IL-1β, IL-18, and IL-6 in supernatants and mice sera was quantified by ELISA using Mouse IL-1 beta/IL-1F2 DuoSet ELISA, Mouse IL-18 DuoSet ELISA, and Mouse IL-6 DuoSet ELISA (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s manual. Fecal lipocalin-2 was quantified by ELISA using Mouse NGAL/Lipocalin-2 ELISA Kit (Beyotime) according to the manufacturer’s instructions.

Six- to 8-week-old mice were intranasally inoculated with the living bacteria or PTx, LOS, and/or Vag8, as described above. On the indicated days after the first inoculation, the mice were euthanized with pentobarbital, and their tracheas were exposed by incising the necks in the median line. A catheter (Surflo Flash 20G; Terumo) was inserted into the exposed tracheas, 0.5 mL of PBS was slowly injected, and the bronchoalveolar lavage fluid (BALF) was thoroughly aspirated. The BALF was centrifuged at 12,000 × g for 5 min, and the supernatant was collected. The concentrations of bradykinin and cytokines in the supernatant were measured with a mouse bradykinin enzyme-linked immunosorbent assay (ELISA) kit (MyBioSource), a mouse IL-1β/IL-1F2 DuoSet ELISA (R&D Systems, code DY401-05), a mouse IL-6 DuoSet ELISA (R&D Systems, code DY406-05), and a mouse TNF-α DuoSet ELISA (R&D Systems, code DY410-05).