At the end of culture, 10μM EdU was added to the media and incubated for 50 min at 37°C. After incubation the media was removed and wells were washed with HBSS. The Matrigel was dissociated with 0.25% Trypsin + 0.5mM EDTA in HBSS for 2 min at 37°C. Excess DMEM-F12 was added to the wells and then harvested to a 50mL conical vial. The organoids were centrifuged and resuspended in 10mL of 3mM EDTA in HBSS (enteroids) or 60mM EDTA in HBSS (colonoids) for 20 min on ice with gentle shaking, then rendered to a single cell suspension with pipetting and filtered through a 40μm filter. Cells were washed twice with HBSS. Viability stain was added at 1:1000 (Fixable viability dye eFluor 660, eBiosciences, cat# 65-0864-14) in HBSS for 30 min on ice in the dark. Cells were then fixed with 4% paraformaldehyde in PBS for 20 min at room temperature in the dark. Following two washes in 1% BSA-PBS cells were concurrently permeabilized (0.5% TritonX100) and stained with Lysozyme antibody (1:10) for 30 min at room temperature [22 (link)]. Cells were washed and then stained with EdU as per the manufacturers’ instructions. Flow cytometric analysis was performed on a Becton Dickinson Fortessa and analyzed by FlowJo vX.0.7 software (Treestar Inc).
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