Time-lapse visualization of PC extrusion after IFN-γ exposure using transgenic reporter organoid lines. BAC transgenic organoids were used that express Lyz2-RFP fusion protein from the Lyz2 genomic region to label PC granules (red signal). Nuclei were labeled using lentivirally expressed H2B-Dendra (green signal). Images were acquired in 30-min intervals for a total of 36(days:hours:minutes:seconds). Shown are confocal images of single Z-planes overlaid to bright field images. Note that despite use of a ubiquitous lentiviral promoter (CMV) and continuous puromycin selection the nuclear fluorescence signal (H2B-Dendra) is most strongly present in differentiated cells: villus enterocytes distal to the budding protrusions and PCs that can be distinguished by the Lyz2-RFP signal. Control small intestinal organoids maintain PC granules and nuclei during the 36 h imaging period.
