To begin culture five times 10 to the fifth DF-1 cells per well in a six well plate with DMEM supplemented with 10%FBS. When the cells reach 80%confluence, discard the serum containing cell culture medium. After washing the cells three times with PBS, add two milliliters of serum-free cell culture medium, followed by IBDV virus solution to each well.
After one hour of virus absorption, wash the cells with PBS three times and incubate them for 24 hours with two milliliters of DMEM supplemented with 2%FBS. Add 500 microliters of trypsin per well, followed by two milliliters of DMEM with 10%FBS after one minute. Centrifuge the cells and remove the supernatant carefully.
After resuspending the cell pellet in PBS, gently vortex the tube for three seconds and centrifuge the cells as described earlier. Using a hemocytometer under a microscope, count the cells to resuspend them in an appropriate volume of flow cytometry staining buffer and vortex the suspension. Add 100 microliters of the single cell suspension into a five milliliter round-bottom polystyrene tube.
Incubate the IBDV infected cells and mock control cells with one microgram of appropriate antibody on ice for 30 minutes in the dark. Gently vortex tube every 10 minutes. Wash the cells with one milliliter of flow cytometry staining buffer and centrifuge the tube.
After discarding the supernatant, resuspend the pellet in 100 microliters of flow cytometry staining buffer. Then incubate the cells with 10 microgram per milliliter of propidium iodide for 10 minutes at room temperature in the dark. And add 400 microliters of flow cytometry staining buffer for the flow cytometry assay.
Perform flow cytometry with the blank control tube followed by the single stained tubes to adjust the voltage and compensation parameters before running all the samples. Flow cytometry indicated that a considerable number of cells were positive for N-terminal fragments of chicken Gasdermin E after IBV infection, whereas the C-terminal fragments of cleaved chicken Gasdermin E were barely detectable by flow cytometry using membrane surface antigen staining. The population of N-terminal fragments of chicken Gasdermin E and propidium iodide double-positive cells in IBDV infected cells was significantly greater than that of mock infected controls, suggesting that this part of IBDV infected cells was undergoing pyroptosis.
