Ax ax r

Three healthy fish were euthanized with an overdose of MS222 (400 mg/L), and their spleens were aseptically collected, homogenized using a 100 µm mesh strainer, and resuspended in FACS media (PBS; 0.1% fetal bovine serum (FBS; Gibco, NY, USA)). Biotinylated chicken IgY anti-salmon IgM (1 mg/mL, Somru BioSciences, Charlottetown, PE, Canada) was labeled with Texas red-avidin (2.5 mg/mL, Thermo Fisher Scientific, MA, USA) by mixing in a 1:1 volume for 30 min at room temperature. Then 50 µL of the spleen cell suspensions were gently mixed with 3 µL of Texas red-IgY anti-salmon IgM, and 1 µL of 4,6-diamidino-2-phenylindole (DAPI, 5 mg/mL concentration) (Thermo Fisher Scientific, MA, USA) in a 1.5 mL centrifuge tube. A 3 µL aliquot of the stained cell suspension was added onto mountain media (Thermo Fisher Scientific, MA, USA) on a microscope slide, covered with a cover slide, and sealed. These samples were visualized by confocal microscope (Nikon AX/AX R, Long Island, NY, USA) for image acquisition. This study was conducted to verify the flow cytometry results.

In all, 2 × 10⁴ PC cells were plated in 8-well chambered cover glass (#C8-1.5H-N, Cellvis). The cells were then fixed with 4% paraformaldehyde followed by incubation with 0.05 mg/ml Filipin III (Sigma, #SAE0087) staining solution at room temperature for 1.5 h. Furthermore, the nuclei were stained with SYTOX Deep Red stain (#P36990, Invitrogen). Fluorescence images of Filipin III staining were taken via a Nikon AX/AX R confocal microscope. The Filipin III staining quantification was analyzed by ImageJ.