Fluorescent Probe Imaging Assay: A Technique to Visualize Oxidative Stress in the Reactive Oxygen Species Inducer-Treated Cultured Intestinal Organoid Cells
Trascrizione
Intestinal organoids recapitulate the gut-like crypt-villus organization having a central lumen comprising dead cells and debris. The lumen is lined by villus-like cells and budding, crypt-like protrusions harboring stem cells.
Different organoid cells produce varying levels of reactive oxygen species, ROS – by-products of metabolic activities, the accumulation of which leads to oxidative stress.
To visualize oxidative stress in varied cells, begin by taking a multi-well plate containing cultured 3D organoids in a basement matrix. The organoid stem cells express a green fluorescence protein or GFP-tagged protein that helps distinguish them during imaging.
Supplement the wells with tert-butyl hydroperoxide – an ROS inducer – and incubate. The inducer activates the membrane-bound NADPH oxidase and stimulates ROS production inside the cells. Stem cells, being metabolically active, produce more ROS than differentiated cells.
Add ROS-sensitive fluorogenic probes. These lipophilic probes rapidly enter the cells and react with ROS to produce red fluorescence. Treat the organoids with Hoechst dye that selectively stains live cell nuclei. Image the organoids using a confocal microscope.
Organoid cells in the periphery with stained nuclei show varying degrees of red fluorescence, which is high in the GFP-expressing stem cells and low in the differentiated cells. The dead cells with inherently high ROS levels exhibit red fluorescence in the lumen.
The intensity of red fluorescence correlates with variable levels of oxidative stress in different cell types.
