HT-29 cells were seeded and grown to 70% confluency on 8-well plates (Lab-Tek, USA). The cells were incubated with 5 μM ICB in media for 2 hours at either 37 °C or 4 °C and 5% CO2 in a humidified incubator. Next, the cells were washed twice with 1XPBS cells and stained with 3 μM Hoechst 33342 for 10 minutes. After a single wash of 1XPBS, the live cells were imaged in Opti-MEM using a Zeiss Axiovert 100 TV epifluorescence microscope under a UV filter (ex: 387/11, em: 447/60) and Cy5.5 filter (ex: 635/40, em: 696/40). For each micrograph, a background subtraction with a rolling ball radius of 50 pixels was applied using ImageJ2 software. After calculating the average mean pixel intensity (MPI) for each micrograph, GraphPad Prism was used to plot the data. For each condition, a total of nine micrographs were analyzed. The experiments were conducted in triplicate.