Alexa fluor 647 phalloidin f actin

F-actin and G-actin measurement was performed as described.⁶⁵ D28 hCOs were dissociated into single cells using the same method used for scRNA-seq. Harvested cells were resuspended in organoid maturation medium and passed through a 40 μm filter strainer to obtain single cells. Cells then seeded at a density of 0.05 million/well on 24-well size coverslips coated with hESC qualified Matrigel. After two days in culture, cells were fixed with 4% PFA for 15 min. Permeabilization and blocking of the samples are performed as mentioned in the section on immunofluorescence microscopy. After blocking, cells were stained with antibody Alexa Fluor™ 647 Phalloidin (F-actin, ThermoFisher Scientific, Cat#A22287), Deoxyribonuclease I, Alexa Fluor™ 488 Conjugate (G-actin, ThermoFisher Scientific, Cat# D12371) and DAPI (ThermoFisher Scientific, Cat#D1306) for 2 h at room temperature. Cells were then washed thrice in 1×PBS and mounted with FluorSave^TM (Merck, Cat#345789) for imaging (Nikon, Eclipse Ti2; Zeiss, Axio Observer Z1 and Nikon, Eclipse TE2000-E). The same exposure settings were used to acquire images from WT and FEZ1-null groups. Images were analyzed with ImageJ/Fiji with quantification details in the following section. At least three independent experiments were performed.