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.