Cytokines level determination in plasma samples
Cytokines in plasma samples were measured on 7th-day post-stress induction with BD CBA Mouse Th1/Th2/Th17 Cytokine Kit (BD Bioscience, San Jose, CA, USA). The kit was used for the simultaneous detection of mouse interferon-γ (IFN-γ), IL-2, IL-4, IL-6, IL-10, IL-17, and TNF-α in a single sample. The operations were performed according to the manufacturer’s instructions. Briefly, beads coated with seven specific capture antibodies were mixed. Subsequently, 50 μL of the mixed captured beads, 50 μL of the unknown serum sample or standard dilutions, and 50 μL of phycoerythrin (PE) detection reagent were added consecutively to each assay tube and incubated for 2 hours at room temperature in the dark. The samples were washed with 1 mL of wash buffer for 5 minutes and centrifuged (200 g). The bead pellet was resuspended in a 300 μL buffer after discarding the supernatant. Samples were measured on the BD Accuri C6 flow cytometer and data analysis was performed with FlowJo software. Individual cytokine concentrations were indicated by their fluorescent intensities. Cytokine standards were serially diluted to facilitate the construction of calibration curves, which were necessary for determining the protein concentrations (pg/mL) of the test samples.