Visualization of Rotavirus-Induced Calcium Signaling in a Human Intestinal Organoid Monolayer

Begin with a monolayer of engineered human intestinal organoid cells expressing a calcium indicator composed of a fluorescent protein, calmodulin, and the M13 peptide.

At low calcium concentrations, the indicator remains in its native, low-fluorescence conformation.

Remove the culture medium and add rotavirus to initiate infection.

Scratch the monolayer with a fine needle to expose the basolateral membrane, the primary site of viral entry.

Incubate the cells to allow viral entry and progression into the early stages of replication.

Discard the medium, wash the monolayer with a buffer to remove unbound virus, and add an imaging medium.

During early replication, infected cells express the viral nonstructural protein 4, that forms calcium-permeable pores in the endoplasmic reticulum, releasing stored calcium.

Calcium leaks into the cytosol and binds to the indicator, triggering a conformational change.

Use fluorescence microscopy to detect the indicator’s bright fluorescent conformation as a readout of virus-induced calcium signaling.

Begin by preparing the virus inoculum. Activate the rotavirus stock by adding 10 micrograms per microliter of Worthington trypsin and incubating at 37 degrees Ce