Cholyl-L-lysine fluorescein disodium

Cholyl-L-lysine fluorescein disodium is a fluorescent substrate used in organoid research.

Guidelines: (The following is our recommended protocol, provided as guidance only and should be adapted according to your specific needs.) 1) Centrifuge the organoids at 300 g for 2 minutes and remove the supernatant. 2) Add 100 µL of media, with or without inhibitors, containing the fluorescent substrate Cholyl-L-lysine fluorescein disodium to each sample and incubate at 37 °C until the designated time point. 3) After incubation, add 1 mL of cold PBS to cease substrate transport and centrifuge at 300 rpm for 2 minutes. 4) Discard the supernatant, wash the samples with 1 mL PBS, and repeat this step three times. Seed the obtained organoids in a 24-well plate containing 300 µL of 37 °C HCM medium, and analyze using an inverted fluorescence microscope and CellSens software. 5) For semi-quantitative analysis, capture at least 7 bright-field images containing a minimum of 7 organoids. Then, capture corresponding fluorescence images for each channel using identical fluorescent settings. Label organoids in bright-field images using Image J software and calculate their area. Next, set an intensity threshold for fluorescence images and maintain it consistent during analysis. Then, determine the fluorescence-positive area using ImageJ and calculate the proportion of the fluorescence-positive area relative to the total organoid area.