Red cell lysis buffer

The spleens were gained on d42 and mononuclear cells were extracted from the spleen of mice using 75-µm cell strainers. Then the red blood cells in samples were cracked with Red Cell Lysis Buffer (biosharp, China), followed by washing three times. After counting, the cells were cultured in RPMI 1640 medium with 10% FBS at a concentration of 1.0 × 10⁷ cells/well. Next, the cells were stimulated in a 5% CO₂ incubator at 37 ℃ for 5 h by using Cell Activation Cocktail (without Brefeldin A) (Biolenged, USA) and Brefeldin A Solution (1000X) (Biolenged, USA) under the guidance of the manufacturer′s instructions. After twice washing, cells were stained with APC/cy7-conjugated Zombie NIR™ (Biolenged, USA) to exclude the dead cells. Regarding surface marker analysis, cells were labeled with percp/cy5.5-conjugated anti-mouse CD3ε and FITC-conjugated anti-mouse CD4⁺. Then cells were washed again, followed by the fixing and permeabilizing operation. Regarding intracellular cytokine marker analysis, cells were stained with BV-conjugated anti-mouse IL-4 and APC-conjugated anti-mouse IFN-γ. The amount of each antibody was added according to the manufacturer′s protocols. Then the cells were washed, followed by the resuspension and staining process. Next, cells were examined by a FACSCalibur flow cytometer (BECKMAN COULTER Cytoflex S, USA) and analyzed with FlowJo10.6.

Hepatocytes were obtained from the liver tissue of mandarin fish. The liver tissue was cut into small pieces and digested with trypsin (Gibco, USA). Tissue was dispersed into cells through cell strainer (Biosharp, China). Red cells were lysed with red cell lysis buffer (Biosharp, China). Cells were cultured with M199 (10% fetal bovine serum, penicillin-streptomycin solution 1‰) (Gibco, USA). The inhibitor of SET1DB chaetocin (17 (link)) (Selleck, USA) was used to treat the cells at a concentration of 3 × 10⁻⁵ mol/L for 17 h, and then the levels of H3K4me3 and pepck mRNA expression were examined with six biological replicates and three technical replicates.