Tuft Cell Profiling in Intestinal Epithelium

For analysis of tuft cells in intestinal epithelium and organoids, cells were surface stained with LIVE/DEAD Fixable Cell stain (ThermoFisher, L34957), and fluorescent conjugated antibodies against EpCAM, CD24, CD45, Siglec-F, and CD90.1 (see Supplementary Table 1 for detailed information, 1:400 in PBS) for 30 minutes. For intracellular staining of CDC42, cells were fixed/permeabilized (ThermoFisher Cat: 00-5521-00), incubated with the anti-CDC42 antibody (ThermoFisher, 1:50) for 1h in room temperature, followed by the anti-rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody (Invitrogen, 1:400) for 1h in 4°C. IECs were defined as live CD4⁻CD8⁻Epcam⁺CD45^low. Successfully transduced organoid cells were defined as CD90.1⁺. Proportion (%) of IECs in the tuft cell lineage were defined as Siglec-F⁺CD24⁺ as previously described^{51 (link)}. Flow cytometry data was analyzed with FlowJo (version 10.8.1).