Legend maxtm elisa kit

IL-6 was measured using LEGEND MAX^TM ELISA kit (BioLegend). The analysis for CCL5/RANTES, CCL2/MCP-1, and IGF-1 was carried out with Quantikine ELISA kit (R&D Systems). All procedures were performed following the manufacturers' instructions. Read-out was performed by dual spectrophotometric measurement: absorbance measured at 570 nm was subtracted from absorbance measured at 450 nm. All samples were assayed in duplicate. On each microplate, a standard curve, obtained from dilution of a standard with known concentration, was included. Concentrations of samples were calculated from the standard curve using a logistic curve-fitting algorithm.

Cardiac γδT cells were enriched from total cardiac cells using anti-PE Microbeads UltraPure (Miltenyi Biotec) after staining with PE-conjugated anti-mouse γδTCR (BD). Isolated cardiac γδT cells were transferred to 96-well Maxisorp plate (Nunc), which was coated overnight with 100 μl of 4-μg/ml anti-CD3 antibody (145-2C11; TONBO Biosciences) and cultured in the presence of 10-ng/ml mIL-1β (Peprotech) and 10-ng/ml mIL-23 (RD systems). After 48 h, the supernatants were collected and assayed for IL-17A production using LEGEND MAX^TM ELISA Kit (BioLegend) according to the manufacturer’s instructions.

Samples of blood (300–500 μL) were taken from the right ventricle of the heart of the halothane-anesthetized animals with a sterile tuberculin syringe. The blood samples were centrifuged at 3000× g for 10 min and then the serum layers were collected and stored at −20 °C until measurement. IL-6 concentrations were determined using the LEGEND MAX^TM Elisa kit (BioLegend Inc., San Diego, CA, USA), according to the instructions of the manufacturer. The sensitivity of the assay was 2 pg/mL. The intraassay coefficient of variation was <5.7%, and the interassay coefficient of variation was <10.7%. Statistical analysis of ELISA data was done using R Studio for Windows (version 1.3.1073). Data are expressed as mean ± SEM, differences were calculated with an ANOVA and a post hoc Bonferroni’s test was performed for significance.