2.4 ELISA
Mice were sacrificed after hypoxic exposure for 7 d. On the 8th day, after blood collection from the mouse orbital vein, the mice were euthanized by cervical dislocation. The colon and spleen of the mice in each group were removed by surgery. The blood samples were allowed to stand at room temperature for 1 hour and processed by centrifugation at 3500 r/min for 15 min, and serum was collected and stored at -80 °C. Double antibody sandwich ELISA kits were used to determine the levels of TNF-α (JM-02415M1, Jing Mei Biotechnology), IL-1β (JM-02465M1, Jing Mei Biotechnology), IFN-γ (JM-02446M1, Jing Mei Biotechnology) and IL-6 (JM-02323M1, Jing Mei Biotechnology) in serum samples and spleen lymphoid specimens. A total of 100 μL of serum was added to the packaged microwell plate and incubated at 37 °C for 90 min. After discarding the supernatant and washing the plate, 100 μl of biotinylated cytokine-specific antibody was added and incubated at 37 °C for 60 min. Then, 100 μL of pro-subsidin HRP was added and incubated at 37 °C for 30 min. The substrate TMB was developed by placing the plate at room temperature for 15 to 20 min. Finally, the stop solution was added, the absorbance was measured at a wavelength of 450 nm by a microplate spectrophotometer.