Peter van Mourik

116 Chapter 4 LIMITATIONS This protocol aims to provide detailed descriptions on how to establish viable human colon organoid cultures and to reproducible FIS results. The quality and fast processing of rectal biopsies is crucial for successful isolation of stem cell crypts. Despite detailed protocols, quality of the colon organoid media can vary between batches and operators and is mainly caused by the variation in self- produced conditioned media like WCM and NCM. Currently, the activity of essential components such as Wnt-3A and Noggin can be assessed in quantitative assays but organoid responses to these factors may not fully be represented by cell-line based reporter assays which can affect the quality of the colon organoid cultures and outcome of FIS results. Therefore, pictures and examples are included in this protocol to provide guidance for the researcher and allow ongoing monitoring of human colon organoid culture quality and to compare FIS data. Various microscopy platforms can be used, but robustness of FIS data is highly dependent on the quality of the fluorescent images, and especially a low signal to noise ratio leads to unreliable data analysis and non-representative results. Therefore, optimized confocal microscopic procedures and efficient imaging software analysis are needed to ensure capturing high-quality images and calculation of FIS AUC.