Through our work we aim to understand how viruses disrupt signaling within the intestinal epithelium. We are particularly interested in understanding the ways in which virus infected cells dysregulate neighboring uninfected cells to contribute to pathogenesis. We've recently discovered that rotavirus infected cells release pulses of ADP, which dysregulates neighboring uninfected cells and facilitates viral spread and exacerbates disease.
While calcium imaging is very popular in fields like neuroscience, it has not been widely used to study epithelial biology or virology. This protocol offers an approach to adapt existing technologies and model systems to study calcium signaling in live, virus-infected epithelial tissue. By engineering organoids to express genetically encoded calcium indicators, our approach allows for consistent, reproducible, long-term imaging in a model system that recapitulates the cellular diversity of the human intestinal epithelium.
We aim to understand the effects of virus-induced paracrine purinergic signaling for both the host and the virus. Our future work will also determine whether this type of signaling is broadly conserved across other viruses and whether the effects are similar. To begin, prepare a one milligram per milliliter collagen IV solution in water and add 95 microliters of this solution to each well of a 10 Well imaging bottom chamber slide.
Incubate the slide at 37 degrees Celsius for 30 minutes to two hours to coat the wells. From the cultured 3D human intestinal organoids, remove WRNE maintenance medium from the wells five to seven days after the last passage. Then, add 500 microliters of 1X PBS containing 0.5 millimolar EDTA per well.
Using a pre-coded one milliliter tip, pipette gently to detach BMM from the plate. Transfer the suspension to a pre-coded 15 milliliter conical tube. Rinse each well with an additional 500 microliters of the PBS/EDTA solution.
Centrifuge the suspension and remove the supernatant and residual BMM. Then, use a pre-coded tip to resuspend the pellet in three milliliters of PBS containing five millimolar EDTA. Centrifuge again and resuspend in two milliliters of enzymatic dissociation buffer.
Incubate the tube at 37 degrees Celsius for five minutes. Then, add three milliliters of CMGF-with 10%FBS and gently mix. Centrifuge and add one milliliter of CMGF-to the pellet.
Vigorously pipette the mixture 80 to 100 times to break the organoids into single cells. Centrifuge and resuspend the pellet in one milliliter of WRNE medium. Obtain a cell count and dilute the cell suspension to achieve 1.25 times 10 to the five cells in 100 microliters.
Next, remove the collagen solution from the previously prepared plate, avoiding touching the bottom of the well. Then, add 100 microliters of the prepared cell solution per well. Incubate for 24 hours in a cell culture incubator at 37 degrees Celsius.
Replace the medium with 100 milliliters of differentiation medium per well after every 24 hours until confluence. After this point, the monolayers are ready for downstream applications. Begin by preparing the virus inoculum.
Activate the rotavirus stock by adding 10 micrograms per microliter of Worthington trypsin and incubating at 37 degrees Celsius for one hour. Then, dilute 50 microliters of the activated viral stock with 50 microliters of CMGF-Next, replace the media from the HIO monolayers with 100 microliters of virus inoculum to infect the monolayers. For rotavirus infection, make a single score across the length of the monolayer using a 25-gauge needle.
Incubate the plates at 37 degrees Celsius for two hours. After incubation, remove the inoculum and wash the monolayers once with 1X PBS. Then, add 100 microliters of phenol red-free differentiation media.
Place the slide in a 37 degree Celsius incubator until ready to image. To begin, set up the epifluorescence microscope. Before imaging, preheat the stage top incubator to 37 degrees Celsius and run humidified carbon dioxide in the chamber at 0.02 liters per minute.
Then, place the slide with infected monolayers into the incubator chamber and seal the lid. Using 20X objective under bright field illumination, select the X and Y coordinates of the fields of view within the monolayer. Optimize imaging parameters and acquire an image using an excitation wavelength of 488 nanometers.
To minimize phototoxicity, set the light source to 50%power with a 50 millisecond exposure time. Ensure that no pixels are saturated and adjust to detect the entire dynamic range of the fluorescent calcium sensor. Set up an imaging loop and acquire an image at every selected X and Y coordinate in a one-minute loop and image this loop continuously.
For fluorescently tagged viruses, acquire an image on the appropriate channel every 10th loop. If using nonfluorescent viruses, perform immunofluorescence staining after imaging. First, fix the monolayers with 100 microliters of formaldehyde.
After incubation, remove the fixative and quench with 100 microliters of 50 millimolar ammonium chloride for 10 minutes. After removing ammonium chloride, permeabilize the monolayers with 100 microliters of 0.1%Triton X-100 for one hour at room temperature. Then remove this solution, add 100 microliters of blocking solution, and incubate for one hour.
Then, remove the blocking solution and add 100 microliters of primary antibodies diluted in 1X PBS. Incubate overnight at four degrees Celsius with gentle rocking. After incubation, remove primary antibodies and wash three times with 1X PBS.
Finally, add 100 microliters of fluorescent conjugated secondary antibodies diluted in 1X PBS. To prevent photobleaching, protect the plate from light and incubate for two hours at room temperature. Remove secondary antibodies after incubation and stain the samples with DAPI solution.
Then, place the fixed monolayers in 100 microliters of 1X PBS for imaging. Place the slide back onto the microscope stage. Reload the X and Y coordinates from the live imaging run and image each multi-point on the channel corresponding to the secondary antibody used for staining.
Reference the images from the live imaging run to ensure capturing the same points. Using this technique, rotavirus infection was imaged in an HIO monolayer after scoring this monolayer. For recombinant strains of rotavirus that express fluorescent proteins such as mRuby, infection was verified during live imaging.
Whereas when a strain that does not express a marker was used, infection was detected by immunofluorescence after imaging.
