Falcon 5 ml round bottom polystyrene test tube

AOs were differentiated using described protocols. On the day of the analysis, organoids were harvested and washed in 10 ml DMEM+P/S. Organoids were dissociated into single cells using TrypLE (Gibco) supplemented with Y‐27632 at 37°C for 30 min. During the single‐cell dissociation, the organoid suspension was vigorously pipetted every 5 min to keep the solution homogenous. Cells were fixed in 4% paraformaldehyde for 2 h at RT. The organoids were permeabilized for 30 min in 0.5% Triton X‐100 (Sigma) and blocked for 30 min in 2% normal goat serum in PBS. Organoids were incubated with primary antibody (mouse‐anti‐acetylated‐α‐tubulin; Santa Cruz; sc‐23950) for 1 h at RT, washed three times with PBS, incubated with secondary antibodies (goat‐anti‐mouse IgG‐Alexa488; Invitrogen) for 1 h at RT and washed two times with PBS. Cells were strained over 35 µm mesh into Falcon® 5 ml Round Bottom Polystyrene Test Tube (Corning; 352235). FACS analysis was performed within 30 min on BD LSR Fortessa X20 4 laser FACS machine. Data analysis was performed using FlowJo (v10.7.2).

The apoptosis assay was performed using the PE Annexin V Apoptosis Detection Kit I (BD Pharmingen™) following the manufacturer's instructions. Briefly, PBLs were incubated for 48 or 72 h at 20°C in 96-well plates with the rIFNs or with media alone. Thereafter, cells were stained with anti-trout IgM [1.14 mAb mouse IgG1 coupled to FITC, 0.425 μg/ml] for 20 min at 4°C. After washing with staining buffer, the cells were centrifuged at 400 × g for 5 min at 4°C, and 1.5 μl of PE Annexin V and 4 μl of 7-aminoactinomycin D (7-AAD) per 200 μl of 1X Annex V Binding Buffer added to the cells. The cells were then incubated for 15 min at room temperature (RT) in the dark before being transferred to flow cytometry tubes (Falcon® 5 mL Round Bottom Polystyrene Test Tube, Corning) containing 400 μl of 1X Annex V Binding Buffer, and analyzed within 1 h on a FACS Celesta flow cytometer.