Isolation and Culture of Kidney-Derived Macrophages from Mudskippers

Kidney-derived MO/MФ from mudskippers were isolated and cultured as reported previously (Guan et al., 2017 (link); Shen et al., 2020 (link)). Briefly, fish kidney leukocyte-enriched fractions were obtained using Ficoll-Hypaque PREMIUM (1.077 g/mL) (GE Healthcare, USA). Non-adherent cells were removed by washing, and adherent cells were incubated in complete medium (RPMI 1640, 5% mudskipper serum, 5% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, USA), 100 U/mL penicillin, 100 μg/mL streptomycin) at 24 °C with 5% CO₂. The HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (Biochrom AG, Germany) supplemented with 10% (v/v) FBS, 100 U/mL penicillin (Sangon Biotech, China), and 100 μg/mL streptomycin (Sangon Biotech) at 37 °C with 5% CO₂. The HEK293T cells were then seeded into 6-well plates to allow growth until 70%–90% confluence on the day of transfection.