Bio plex pro mouse cytokine th1 th2 assay

After euthanasia and exposure of the trachea, a Safelet Cath (22 g × 1″, Exel) was inserted into trachea and BAL was performed using 1 ml PBS pH 7.4. The lavage sample was centrifuged at 2,500 × g for 5 min at 4°C to pellet the cells and the supernatant was used for cytokine analysis. Both Th-1 and Th-2 cytokines were analyzed using a Bio-Plex Pro™ Mouse Cytokine Th1/Th2 Assay and Bio-Plex Pro™ Mouse Cytokine 23-plex Assay (Bio-Rad). IL-8 was quantified in BAL fluid using the Mouse IL-8 ELISA kit (MyBioSource) according to the manufacturer’s instructions. Briefly, a 48-well plate was coated with the primary capture Ab and stored overnight at 4°C. Following washes with PBS-T, the plate was blocked with assay diluent for 1 h at room temperature. Detection Ab and diluted Avidin-HRP were then added to the plate, with washes between additions, and the plate was developed with TMB substrate in the dark. Absorbance was then measured at 450 nm.

Skin samples were homogenized in PBS containing 0.1% Tween 20 (Nacalai Tesque) and 1% Protease Inhibitor Cocktail Set III (Merck) and centrifuged. Blood samples were collected under anaesthesia, and serum was isolated after coagulation and centrifugation. The concentrations of IFN‐α, heat shock protein 70 (HSP70) and IL‐18 were measured by enzyme‐linked immunosorbent assay (ELISA) using VeriKine‐HS Mouse IFN‐α All Subtype ELISA Kit (PBL Assay Science), Mouse HSP70 ELISA kit (CUSABIO) and Mouse IL‐18 SimpleStep ELISA kit (Abcam). The concentrations of IL‐1β, IFN‐γ, tumor necrosis factor (TNF)‐α and IL‐17A were measured using Bio‐Plex Multiplex Immunoassay System (Bio‐Rad) with Bio‐Plex Pro Mouse Cytokine IL‐1β Set and Bio‐Plex Pro Mouse Cytokine Th1/Th2 Assay (Bio‐Rad).

The Bio-Plex Pro mouse cytokine Th1/Th2 assay (Bio-Rad, Hercules, CA, USA) was used to quantify cytokines including IL-2, IL-4, IL-5, IL-10, IL-12p70, TNF-α, interferon (IFN)-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Lung homogenate samples were diluted 1:2 with the Bio-Plex sample diluent. Standard dilution was performed using the transport medium. Beads were dispensed into wells of a 96-well plate, and the samples, standards, and blanks were added and vortexed for 30 min. After washing with Wash Buffer, a secondary antibody was added to the wells, and the plate was vortexed for 30 min. The wells were washed again, phycoerythrin-conjugated streptavidin was added, and the plate was vortexed for 10 min. After washing again, Assay Buffer was added, and measurement were carried out by Luminex 200 (Merck, Kenilworth, NJ, USA). The concentrations of cytokines (pg/mL) were determined using the Bio-Plex Manager Software (Bio-Rad).

Bio-Plex Pro Mouse Cytokine T_H1/T_H2 Assay (Bio-Rad, catalog no. m6000003j7) was performed according to the manufacturer’s guidelines. Cells (2.5 × 10⁵) isolated from the ischemic thighs of mice were cultured with splenocytes (2.5 × 10⁵) isolated from naïve mice in serum-free AIM V medium in the presence of OVA protein (20 μg/ml) or ct26-gp70 peptide (20 μg/ml) as an irrelevant peptide control. After 3 days of culture, media supernatants were collected and analyzed on a BioPlex 3D System (Bio-Rad). To determine OVA-specific cytokine production, the concentration of cytokines detected in the ct26-gp70 peptide condition was subtracted from the OVA protein condition.