Name: Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators (Video) | JoVE Journal
Uploaded: 2024-01-19T14:05:00.000+00:00
Duration: 8 min 1 s
Description: Learn live calcium imaging in virus-infected human intestinal organoids using genetically encoded calcium indicators. This method reveals how rotavirus disrupts epithelial signaling and dysregulates neighboring cells.

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5 minutes

This work was supported by grants R01DK115507 and R01AI158683 (PI: J. M. Hyser) from the National Institutes of Health (NIH). Trainee support was provided by NIH grants F30DK131828 (PI: J.T. Gebert), F31DK132942 (PI: F. J. Scribano), and F32DK130288 (PI: K.A. Engevik). We would like to thank the Texas Medical Center Digestive Diseases Enteroid Core for providing the organoid maintenance media.

All of the human intestinal organoids (HIOs) used in this protocol and the representative experiments were derived from human tissue obtained and maintained by the Texas Medical Center Digestive Diseases Enteroid Core. All samples were collected in accordance with a protocol approved by the Institutional Review Board at Baylor College of Medicine.
1. Preparation of materials and reagents
<ol>
	<li>For organoid maintenance, gather cell-culture treated 24-well plates, basement membrane matrix (BMM), 15 mL conical tubes, and 1.5 mL conical tubes. 
	<ol>
		<li>To prepare complete media without growth factors (CMGF-), to 500 mL of advanced DMEM F12 add 5 mL of 1M HEPES, 5 mL of 100x antibiotic-antimycotic, 5 mL of 100x glutamine supplement. </li>
		<li>To prepare Wnt-, R-spondin-, and Noggin-containing (WRNE) media, mix equal parts CMGF- and Wnt-conditioned media, add Noggin-conditioned medium, 10% by volume, R-spondin conditioned medium, 20% by volume, 50 ng/mL human epidermal growth factor, 10 mM nicotinamide, 10 nM [Leu15]-Gastrin I, 500 nM A-83-01, 10 µM SB202190, 1x B27 supplement, 1x N2 supplement, and 1 mM N-acetylcysteine.</li>
	</ol>
	</li>
	<li>For lentiviral transduction, prepare Fetal Bovine Serum (FBS), 0.05% Trypsin-EDTA in 1x phosphate buffered saline (PBS), CMGF- with 10% FBS, Sterile 1x PBS, Polybrene, 10 µM Y-27632, Lentivirus, High Wnt WRNE + 10 µM Y-27632</li>
	<li>For generating organoid monolayers, prepare glass bottom 10-well cell culture slide, FBS, Collagen IV (1 mg/mL in de-ionized (di)H2O), basement membrane matrix, 0.5 mM EDTA in 1x PBS, 5 mM EDTA in 1x PBS, enzymatic dissociation buffer, CMGF- with 10% FBS, WRNE + 10 µM Y-27632.</li>
	<li>For viral infection of organoid monolayers, prepare trypsin, rotavirus stock, CMGF-, 25G needle, Sterile 1x PBS, and phenol red-free differentiation media.
	<ol>
		<li>To prepare phenol red-free differentiation media, take 500 mL of phenol red-free cell culture media, add 5 mL of 100x MEM non-essential amino acids, 5 mL of 100x L-Glutamine, 5 mL of 100 nM sodium pyruvate, and 7.5 mL of 1M HEPES.</li>
	</ol>
	</li>
	<li>For immunofluorescence staining of organoids, prepare 4% formaldehyde (16% formaldehyde diluted in 1x PBS), Triton X-100 (0.1% Triton X-100 in 1x PBS), Bovine serum albumin (3% bovine serum albumin in 1x PBS), NH4Cl solution (50 mM), DAPI (1 µg/mL DAPI solution in 1x PBS).</li>
</ol>
2. Engineering organoids to express genetically encoded calcium sensors
NOTE: This protocol describes the steps to transduce a single well of 3-dimensional human intestinal organoids plated in 30 μL of Basement Membrane Matrix (BMM) on a 24-well plate13. Most lines will contain around 400,000 cells per well. A second, non-transduced well should be included as a control. Keep all reagents and cell suspensions on ice.
<ol>
	<li>After 2-5 days from the last passage, remove maintenance WRNE medium from two wells of HIOs. Replace with 300 μL of 0.05% Trypsin-EDTA per well and gently pipette up and down 5x to detach the BMM from the plate. Place in a 37 °C incubator for 4 min.</li>
	<li>Add 500 μL of CMGF- + 10% FBS per well. Pre-coat a 1 mL low-bind pipette tip with FBS by pipetting 1 mL of FBS up and down 2x. FBS can be re-used on multiple tips. With the pre-coated tip, pipette the HIOs up and down 10x.</li>
	<li>Transfer the contents of each well to its own pre-coated 1.5 mL microcentrifuge tube. Rinse each well with an additional 500 μL of CMGF- and add the wash to the respective tube.</li>
	<li>Centrifuge the tubes at 100 x g in a swinging bucket centrifuge for 5 min at 4 °C. Remove supernatant and any residual BMM.</li>
	<li>Resuspend in 1 mL of 1x PBS. Split each tube into two microcentrifuge tubes for 4 total tubes. Centrifuge the tubes at 100 x g for 5 min at 4 °C. Remove supernatant.</li>
	<li>Resuspend each tube one additional time in 1x PBS and centrifuge at 100 x g for 5 min at 4 °C.</li>
	<li>Prepare 400 µL of transduction medium and control medium (Table 1). Resuspend 2 tubes in 200 µL of the control medium and 2 tubes in 200 µL of the transduction medium.</li>
	<li>Incubate in a 37 °C cell culture incubator for 24 h. Resuspend by pipetting up and down with a coated tip periodically (e.g., 2 h post transduction (hpt), 12 hpt, 18 hpt) to encourage uniform transduction.</li>
	<li>After 24 h, centrifuge tubes at 100 x g for 5 min at 4 °C. Remove supernatant.</li>
	<li>Resuspend pellet in 500 µL of 1x PBS to wash. Centrifuge at 100 x g for 5 min at 4 °C. Remove supernatant.</li>
	<li>Using an ice-cold 200 µL pipette tip, resuspend each of the 4 pellets in 30 µL of BMM. Gently pipette up and down to disperse evenly.</li>
	<li>Plate the contents of each tube into its own well on a 24-well plate. Incubate for 10 min at 37 °C to allow BMM to solidify before adding 500 μL of HighWnt WRNE + 10μM Y-27632.</li>
	<li>Allow transduced HIOs to grow for 1 week, refreshing the media (HighWnt WRNE + 10 μM Y-27632) every other day. After 1 week, check for the expression of the fluorescent indicator by microscopy. If the signal is strong, begin drug selection. If the signal is weak, repeat the transduction as described above.</li>
	<li>Once the line is established, verify the function of the fluorescence indicator via agonist treatment. 100nM ADP is a reliable agonist for GCaMP validation. Test 3D organoids for initial validation as described below.
	<ol>
		<li>After a passage, plate a well (or multiple) of organoids in BMM on a separate imaging-bottom plate. Plate the organoids at about 1/3 of the normal density to avoid excess overlap when imaging. Do not continue to passage these HIOs following agonist treatment, as it is difficult to ensure sterility.</li>
		<li>Allow 2-3 days for the HIOs to recover post-passage. Before imaging, switch the media to phenol red-free differentiation media.</li>
		<li>Using a fluorescent microscope, set up a 3 min run with images acquired every 5 s using 488 nm excitation and a FITC/GFP filter set. After 30 s of imaging, add 100 nM ADP or vehicle control. Continue imaging until the signal returns to near-baseline, ~2 min. A transient, ~2-fold increase in GCaMP fluorescence with ADP treatment indicates successful transduction and biosensor function. For more precise estimate of transduction efficiency, repeat agonist test with imaging using monolayers generated via the process described in part 3.</li>
	</ol>
	</li>
</ol>
3. Preparation of HIO monolayers for live fluorescence imaging
<ol>
	<li>Coat all wells of a 10-well imaging bottom chamber slide with Collagen IV. To do this, mix 34 μL of 1 mg/mL Collagen IV with 960 μL of sterile deionized water. Add 95 μL of diluted collagen IV solution to each well and incubate at 37 °C for 0.5-2 h.</li>
	<li>Remove the WRNE maintenance medium from 4 wells of 3D HIOs. HIOs should be 5-7 days from their last passage. 
	NOTE: 1 10-well plate typically requires ~1.25 x 106 cells. This typically requires 2-4 wells of 3D HIOs plated in 30 μL of BMM each, but will vary based on density. </li>
	<li>Add 500 μL of 1x PBS + 0.5 mM EDTA per well. With a pre-coated 1 mL tip, pipette up and down gently to detach BMM from the plate. Transfer the suspension to a pre-coated 15 mL conical tube, combining like wells into the same tube.</li>
	<li>Rinse each well with an additional 500 μL of PBS + 0.5 mM EDTA. Centrifuge at 300 x g for 5 min at 4 °C. Remove the supernatant and residual BMM.</li>
	<li>Using a pre-coated tip, resuspend the remaining pellet in 3 mL of PBS + 5 mM EDTA (note that this is 10x more EDTA than the first wash).</li>
	<li>Centrifuge at 300 x g for 5 min at 4 °C. Remove the supernatant and residual BMM. Resuspend the pellet in 2 mL of enzymatic dissociation buffer.</li>
	<li>Incubate in 37 °C bead/water bath for 5 min. Add 3 mL of CMGF- + 10% FBS and pipette gently to mix.</li>
	<li>Centrifuge at 300 x g for 5 min at 4 °C. Remove the supernatant. Add 1 mL of CMGF-.</li>
	<li>Vigorously pipette up and down with a pre-coated tip 80x-100x to mechanically break HIOs into single cells. Centrifuge at 300 x g for 5 min at 4 °C. Remove supernatant.</li>
	<li>Resuspend in 1 mL of WRNE + 10 µM Y-27632. Obtain a cell count for the 1 mL suspension.</li>
	<li>Dilute the cell suspension with WRNE + 10µM Y-27632 to achieve a concentration of 1.25 x 105 cells/100 μL (1.25 x 106 cells/mL).</li>
	<li>Using a 200 μL pipette, remove the collagen solution from the plate prepared in step 1, as the collagen has now settled to the bottom of the well. Avoid touching the bottom of the well with the pipette tip.</li>
	<li>Using a pre-coated 200 μL pipette tip, add 100 μL of the cell solution from step 3.11 (1.25 x 105 cells) per well.</li>
	<li>Incubate for 24 h in a 37 °C cell culture incubator. After 24 h, remove the culture medium from all wells and replace it with 100 μL of differentiation medium per well. 
	NOTE: By this point, the cells should be adhering to the plate. Note that the monolayer likely will not be confluent or fully planar.</li>
	<li>Place the slide back into the 37 °C cell culture incubator. Refresh the differentiation medium every 24 h until the monolayer is confluent. This usually requires 3-5 days from plating. After this point, the monolayers are ready for downstream applications.</li>
</ol>
4. Viral infection of HIO monolayers
<ol>
	<li>Prepare the virus inoculum. If necessary, trypsin activate the virus stock. For rotavirus, add 10 µg/mL Worthington's Trypsin to the virus stocks and incubate at 37 °C for 1 h.
	<ol>
		<li>Dilute the activated virus stock using one of the two following methods.
		<ol>
			<li>If aiming for the maximum number of infected cells using only one viral strain, mix 50 μL of the activated viral stock with 50 μL CMGF-.</li>
			<li>If comparing multiple strains, prepare the inoculums to achieve equivalent multiplicities of infection (MOIs). Use the following formula to determine the volume of virus stock per well necessary for the desired MOI. Add CMGF- to the volume of virus stock calculated to achieve a final volume of 100 μL per well. 
			<img alt="Viral titer calculation formula, MOI application, virology experiment, equation diagram." src="/files/ftp_upload/66132/66132eq01.jpg" style="margin: auto;" data-altupdatedat="1787630408000" data-alt="Equation 1"> 
			NOTE: A single HIO monolayer plated in a well with a diameter of 5.46 mm (96-well plate) contains about 200,000 cells. Because of the poor efficiency of infection in monolayers, a high MOI (100-1 million viral particles per cell) is preferred. The optimal MOI must be determined empirically.</li>
		</ol>
		</li>
		<li>Infect the monolayers by gently removing the media from the HIO monolayer using a 200 mL pipette. Replace with 100 mL of virus inoculum or mock inoculum.
		<ol>
			<li>Since most rotavirus stocks are propagated in MA104 cells, use an uninfected MA104 cell lysate for the mock inoculum. Use a volume equivalent to the volume of virus stock used for the infected wells and add CMGF- for a final volume of 100 mL per well.</li>
			<li>For viruses that infect cells basolaterally, such as rotavirus14, use a 25G needle to score the monolayer. It is easiest to make a single score across the length of the monolayer, from the bottom to the top of the well. Gently press the needle into the monolayer at an angle with the bevel side up, opposite the direction of motion, and drag it across the length of the monolayer to create a scar (Figure 2A).</li>
		</ol>
		</li>
		<li>Incubate in a 37 °C incubator for 2 h. Remove the virus and mock inoculum. Wash monolayers once with 1x PBS.</li>
		<li>Add 100 μL of phenol red-free differentiation media. Place slide in a 37 °C incubator until ready to image. The optimal imaging window will vary based on the kinetics of the viral infection. For rotavirus, begin imaging at 6-8 h post infection.</li>
	</ol>
	</li>
</ol>
5. Ca2+ Imaging of infected monolayers
<ol>
	<li>Pre-heat the stage-top incubator to 37 °C. Run humidified CO2 at 0.02 L/min. Place slide with the infected monolayers into the incubator chamber and seal the lid.</li>
	<li>Using brightfield (BF) illumination and a 20x objective, select the X and Y coordinates of the fields of view within the monolayer that will be imaged. It is best to select points with the scored region in view, as most infected cells will be near the scratch.</li>
	<li>Optimize imaging parameters and acquire an image using 488 nm excitation. To minimize phototoxicity, set the light source to 50% power with a 50 ms exposure time. The appropriate acquisition parameters may vary and should be optimized by acquiring multiple acquisitions. Ensure no pixels are saturated. Adjust exposure time and light power as needed to ensure the entire dynamic range of the fluorescent calcium sensor can be detected.</li>
	<li>If using other fluorophores (e.g., fluorescently tagged viruses), repeat optimization on these channels. Set up the imaging loop and acquire a 488 nm image at every selected X, Y coordinate in a 1 min loop. Image this loop continuously. When using a fluorescently tagged virus, acquire an image on the appropriate channel every 10th loop (i.e., 1 image/10 min).</li>
	<li>Collect images for ~18 h. If using viruses without fluorescent tags, fix monolayers and perform immunofluorescence staining to identify infected cells. Perform this after completing the imaging run as described below.
	<ol>
		<li>Remove imaging media and replace with 100 μL of 4% formaldehyde in 1x PBS. Incubate at room temperature for 30 min.</li>
		<li>Remove fixative and quench with 100 μL of 50mM NH4Cl for 10 min. Remove NH4Cl. Permeabilize the monolayers with 100 μL of 0.1% Triton X-100 in 1x PBS for 1 h at room temperature or overnight at 4 °C.</li>
		<li>Remove permeabilization buffer. Block with 100 μL of 3% bovine serum albumin in 1x PBS for 1 h.</li>
		<li>Remove the blocking solution. Add primary antibodies diluted to the recommended/optimized concentration in 1x PBS. Add 100 μL of antibody solution per well.</li>
		<li>Incubate overnight at 4 °C with gentle rocking. Remove primary antibodies. Wash 3x with 1x PBS.</li>
		<li>Add fluorescent-conjugated secondary antibodies diluted to the recommended/optimized concentration in 1x PBS. Add 100 μL per well. Incubate for 2 h at room temperature. 
		NOTE: FPBase15 is a great resource to help select secondaries that will avoid bleed-through when multiplexing. Most secondaries are effective at a 1:1000 dilution in 1x PBS.</li>
		<li>Remove secondary antibodies. Make a 1 µg/mL DAPI solution in 1x PBS and add 100 μL per well. Incubate for 20 min. Remove DAPI. Wash 3x with 1x PBS.</li>
		<li>Keep fixed monolayers in 100 μL 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 that corresponds to the secondary antibody that was used for staining. Reference the images from the live imaging run to ensure the same points are captured.</li>
	</ol>
	</li>
</ol>
6. Quantitation of intercellular calcium waves
<ol>
	<li>Ensure Fiji is Just ImageJ (FIJI) is installed with the following plug-ins: Bio-Formats (should be pre-installed in FIJI but will not be in ImageJ); Cookbook: To install, from the FIJI menu, go to Help &gt; Updates &gt; Manage update sites. Check Cookbook. Restart FIJI.</li>
	<li>Split the data file from the live imaging run into multiple files, separating each X, Y coordinate. In Nikon NES Elements, select File &gt; Import/Export &gt; Split Multipoints. Select a folder for export and an appropriate prefix.</li>
	<li>Open FIJI. Load a single file from the split multipoints. If using a multi-channel image, split the channels. Select the window with the images from the 488 nm channel (GCaMP).</li>
	<li>Run the DeltaF Up function to determine the change in each pixel value from one timepoint to the next. To do this, select Cookbook &gt; T-functions &gt; Delta F Up.</li>
	<li>Scroll through the stack until an image with a calcium wave is found. With the calcium wave in view, set a threshold by clicking Image &gt; Adjust &gt; Threshold. Adjust the lower limit to minimize the amount of signal that is not part of the wave that makes it past the threshold. For 16-bit images acquired using the parameters described above, start with a lower threshold of 600 and adjust as needed.</li>
	<li>Run the particle analyzer to segment waves by clicking Analyze &gt; Analyze Particles. Set the minimum size of wave in µm2 by adjusting the range. It is set to 0-infinity at baseline. For images of MA104 cells acquired using a 20x objective, start by increasing the lower limit to 10,000 µm2 and adjust as appropriate.</li>
	<li>Check the boxes for Display results, and Clear results. From the "Show" dropdown menu, select Outlines, then click OK. This should result in 2 outputs: One window, Results, will list each wave detected, the area of the wave, and its mean, minimum, and maximum intensity values (based on delta F). The other window, titled Drawing of [file name], will include a new stack with the outlines of segmented waves. Use this to verify wave detection. Adjust threshold values and size range if needed, and re-check the wave calls.</li>
	<li>For batch processing, use the macro script included (Supplementary Coding File 1). To utilize this, follow the steps described below.
	<ol>
		<li>Split multi-points into single files as described in step 6.2. Open FIJI. Select Process &gt; Batch &gt; Macro. In the "input" box, provide the map to the folder containing the split multipoint files. Leave the "output" box empty.</li>
		<li>Paste the script from Supplementary Coding File 1 into the box. Adjust the intensity threshold and size threshold in the script to reflect the optimized parameters from steps 6.5 and 6.6.</li>
		<li>Click Process. Depending on the number of multi-points and processing speed of the computer, it can take 30 min or longer to process all images. Once complete, the data will be written into a new spreadsheet file named "Rename me after writing" on the Desktop. The output file should contain, for each multipoint, a wave count and wave metrics (area, mean intensity, minimum and maximum intensity, and intensity density).</li>
	</ol>
	</li>
</ol>

Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers

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<li>Mroz, M. S., Keely, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidermal+growth+factor+chronically+upregulates+Ca2%2B-dependent+Cl%E2%88%92+conductance+and+TMEM16A+expression+in+intestinal+epithelial+cells%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Epidermal growth factor chronically upregulates Ca2+-dependent Cl− conductance and TMEM16A expression in intestinal epithelial cells.</a> J Physiol. 590 (8), 1907-1920 (2012).</li>
<li>Sui, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dual+role+of+Ca2%2B-activated+Cl%E2%88%92+channel+transmembrane+member+16A+in+lipopolysaccharide-induced+intestinal+epithelial+barrier+dysfunction+in+vitro%5BTitle%5D)+AND+%22Cell+Death+Dis%22%5BJournal%5D)">Dual role of Ca2+-activated Cl− channel transmembrane member 16A in lipopolysaccharide-induced intestinal epithelial barrier dysfunction in vitro.</a> Cell Death Dis. 11 (5), 404(2020).</li>
<li>Bellono, N. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enterochromaffin+cells+are+gut+chemosensors+that+couple+to+sensory+neural+pathways%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Enterochromaffin cells are gut chemosensors that couple to sensory neural pathways.</a> Cell. 170 (1), 185-198.e16 (2017).</li>
<li>Paradis, T., Bègue, H., Basmaciyan, L., Dalle, F., Bon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tight+junctions+as+a+key+for+pathogens+invasion+in+intestinal+epithelial+cells%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Tight junctions as a key for pathogens invasion in intestinal epithelial cells.</a> Int J Mol Sci. 22 (5), 2506(2021).</li>
<li>Samak, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium%2FAsk1%2FMKK7%2FJNK2%2Fc-Src+signalling+cascade+mediates+disruption+of+intestinal+epithelial+tight+junctions+by+dextran+sulfate+sodium%5BTitle%5D)+AND+%22Biochem+J%22%5BJournal%5D)">Calcium/Ask1/MKK7/JNK2/c-Src signalling cascade mediates disruption of intestinal epithelial tight junctions by dextran sulfate sodium.</a> Biochem J. 465 (3), 503-515 (2015).</li>
<li>Deng, H., Gerencser, A. A., Jasper, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Signal+integration+by+Ca2%2B+regulates+intestinal+stem+cell+activity%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Signal integration by Ca2+ regulates intestinal stem cell activity.</a> Nature. 528 (7581), 212-217 (2015).</li>
<li>Saurav, S., Tanwar, J., Ahuja, K., Motiani, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dysregulation+of+host+cell+calcium+signaling+during+viral+infections%3A+Emerging+paradigm+with+high+clinical+relevance%5BTitle%5D)+AND+%22Mol+Aspects+Med%22%5BJournal%5D)">Dysregulation of host cell calcium signaling during viral infections: Emerging paradigm with high clinical relevance.</a> Mol Aspects Med. 81, 101004(2021).</li>
<li>Chang-Graham, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rotavirus+calcium+dysregulation+manifests+as+dynamic+calcium+signaling+in+the+cytoplasm+and+endoplasmic+reticulum%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Rotavirus calcium dysregulation manifests as dynamic calcium signaling in the cytoplasm and endoplasmic reticulum.</a> Sci Rep. 9 (1), 10822(2019).</li>
<li>Hyser, J. M., Collinson-Pautz, M. R., Utama, B., Estes, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rotavirus+disrupts+calcium+homeostasis+by+NSP4+viroporin+activity%5BTitle%5D)+AND+%22mBio%22%5BJournal%5D)">Rotavirus disrupts calcium homeostasis by NSP4 viroporin activity.</a> mBio. 1 (5), e00265-e00310 (2010).</li>
<li>Pham, T., Perry, J. L., Dosey, T. L., Delcour, A. H., Hyser, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Rotavirus+NSP4+viroporin+domain+is+a+calcium-conducting+ion+channel%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">The Rotavirus NSP4 viroporin domain is a calcium-conducting ion channel.</a> Sci Rep. 7, 43487(2017).</li>
<li>Crawford, S. E., Hyser, J. M., Utama, B., Estes, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autophagy+hijacked+through+viroporin-activated+calcium%2Fcalmodulin-dependent+kinase+kinase-%CE%B2+signaling+is+required+for+rotavirus+replication%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Autophagy hijacked through viroporin-activated calcium/calmodulin-dependent kinase kinase-β signaling is required for rotavirus replication.</a> Proc Natl Acad Sci U S A. 109 (50), E3405-E3413 (2012).</li>
<li>Crawford, S. E., Criglar, J. M., Liu, Z., Broughman, J. R., Estes, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((COPII+vesicle+transport+is+required+for+Rotavirus+NSP4+interaction+with+the+autophagy+protein+LC3+II+and+trafficking+to+viroplasms%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">COPII vesicle transport is required for Rotavirus NSP4 interaction with the autophagy protein LC3 II and trafficking to viroplasms.</a> J Virol. 94 (1), e01341(2019).</li>
<li>Pando, V., Iša, P., Arias, C. F., Ló Pez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+calcium+on+the+early+steps+of+Rotavirus+infection%5BTitle%5D)+AND+%22Virology%22%5BJournal%5D)">Influence of calcium on the early steps of Rotavirus infection.</a> Virology. 295 (1), 190-200 (2002).</li>
<li>Hyser, J. M., Estes, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathophysiological+consequences+of+calcium-conducting+viroporins%5BTitle%5D)+AND+%22Annu+Rev+Virol%22%5BJournal%5D)">Pathophysiological consequences of calcium-conducting viroporins.</a> Annu Rev Virol. 2 (1), 473-496 (2015).</li>
<li>Strtak, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recovirus+NS1-2+has+viroporin+activity+that+induces+aberrant+cellular+calcium+signaling+to+facilitate+virus+replication%5BTitle%5D)+AND+%22mSphere%22%5BJournal%5D)">Recovirus NS1-2 has viroporin activity that induces aberrant cellular calcium signaling to facilitate virus replication.</a> mSphere. 4 (5), e00506-e00519 (2019).</li>
<li>In, J. G., Foulke-Abel, J., Clarke, E., Kovbasnjuk, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+colonoid+monolayers+to+study+interactions+between+pathogens%2C+commensals%2C+and+host+intestinal+epithelium%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Human colonoid monolayers to study interactions between pathogens, commensals, and host intestinal epithelium.</a> J Vis Exp. (146), 59357(2019).</li>
<li>Hirota, A., AlMusawi, S., Nateri, A. S., Ordóñez-Morán, P., Imajo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biomaterials+for+intestinal+organoid+technology+and+personalized+disease+modeling%5BTitle%5D)+AND+%22Acta+Biomater%22%5BJournal%5D)">Biomaterials for intestinal organoid technology and personalized disease modeling.</a> Acta Biomater. 132, 272-287 (2021).</li>
<li>Cevallos Porta, D., López, S., Arias, C. F., Isa, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polarized+rotavirus+entry+and+release+from+differentiated+small+intestinal+cells%5BTitle%5D)+AND+%22Virology%22%5BJournal%5D)">Polarized rotavirus entry and release from differentiated small intestinal cells.</a> Virology. 499, 65-71 (2016).</li>
<li>Mirabelli, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+Norovirus+efficiently+replicates+in+differentiated+3D-human+intestinal+enteroids%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Human Norovirus efficiently replicates in differentiated 3D-human intestinal enteroids.</a> J Virol. 96 (22), e0085522(2022).</li>
<li>Icha, J., Weber, M., Waters, J. C., Norden, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phototoxicity+in+live+fluorescence+microscopy%2C+and+how+to+avoid+it%5BTitle%5D)+AND+%22Bioessays%22%5BJournal%5D)">Phototoxicity in live fluorescence microscopy, and how to avoid it.</a> Bioessays. 39 (8), 28749075(2017).</li>
<li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+of+NEMO+as+calcium+indicators+with+large+dynamics+and+high+sensitivity%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Engineering of NEMO as calcium indicators with large dynamics and high sensitivity.</a> Nat Methods. 20 (6), 918-924 (2023).</li>
</ol>

The authors have no competing financial interests to disclose.

Alterations in cytosolic Ca2+ levels can be both a cause and effect of pathologies within the epithelium10,16,17. Increases in cytosolic calcium can directly drive secretion via activation of the calcium-dependent chloride channel TMEM16A18,19. Activation of TMEM16A in response to Ca2+ allows for the apical efflux of chloride, establishing an osmotic gradient that promotes fluid secretion20,21. Furthermore, increases in Ca2+ within enteroendocrine cells trigger the release of serotonin, which stimulates afferent sensory neurons that can modulate activity within the enteric nervous system22. Ca2+ signals can also modulate tight junctions to influence barrier function23,24, and regulate proliferation in intestinal stem cells25. Thus, dysregulation of Ca2+ can have broad and far-reaching effects.
Many viruses manipulate cytosolic Ca2+ to create a cellular environment conducive to replication26. Rotavirus is a prime example of this within the intestinal epithelium27. Rotavirus non-structural protein 4 (NSP4) is a Ca2+-conducting channel that allows for the efflux of Ca2+ from the endoplasmic reticulum into the cytosol28,29. This stimulates autophagy and aids the assembly of the outer capsid30,31,32. Rotavirus infection also induces the release of ADP, triggering intercellular Ca2+ waves that contribute to pathogenesis17. Other enteric viruses, including both caliciviruses and enteroviruses, encode Ca2+-conducting viroporins that trigger similar dysregulation of Ca2+ within infected cells33,34.
Live-cell Ca2+ imaging of human intestinal organoids offers a versatile platform to study virus-induced signaling within the epithelium. However, the strength of the conclusions drawn from the imaging data depends, inextricably, on the health and quality of the HIOs, appropriate imaging parameters, and a sound analytical approach.
Efficient lentiviral transduction is often the limiting step when establishing new HIO lines, but high-titer lentivirus and multiple transductions help to ensure sufficient transgene expression. Importantly, this will have to be balanced with the potential for significant cellular stress upon transduction. This is most often temporary and subsides after selection and multiple passages. Nonetheless, it is critical that HIOs engineered to express GECIs display normal growth and proliferation before beginning experiments. Likewise, dispersing 3-dimensional HIOs and reforming them as monolayers induces cellular stress. It is best practice to allow three or more days for monolayers to adapt after plating. Coating the plates with collagen IV is critical for monolayer integrity35,36. Aliquoting collagen IV and storing at -80 &#176;C&#160;between uses helps to ensure monolayer adherence. Determining the optimal seeding density for each HIO line can also help to improve consistency. Finally, including the proper controls, and comparing them across experiments, is indispensable to ensure any observation represents a response to the variable in question, rather than a technical confounder.
While scoring a monolayer can cause unwanted cell damage, it greatly enhances RV infection of 2D HIOs. Figure 2 illustrates this, as almost all infected cells are directly adjacent to the scratch. Experimental evidence suggests that RV infects most efficiently at the basolateral surface of intestinal epithelial cells37. Thus, it is predicted that scoring a monolayer enhances infectivity by exposing the basolateral surface. Efficient monolayer infection can also be achieved by plating HIOs on polycarbonate membranes suspended in wells containing culture medium, which allow access to the basolateral surface. However, the relatively long distance between the plate and the polycarbonate membrane, the polycarbonate membrane itself, and the plastic surface at the base of the well render them inappropriate for epifluorescence imaging. Thus, the scoring method described in this protocol remains the favored approach. Given that scoring itself may lead to signaling perturbations, the inclusion of the uninfected, scored control is critical for interpretation. For viruses, such as human norovirus, that efficiently infect via the apical membrane, scoring the monolayer is not required38.
As with any live imaging system, phototoxicity can easily confound experiments utilizing HIO monolayers. Throughout an imaging experiment, it is important to monitor for unexpected vacuolization, membrane blebbing, or other morphological indicators of stress39. If any of these signs are detected, the duration, frequency, or power of excitation must be reduced. Increasing the signal-to-noise ratio often comes at the expense of long-term cell health thus, empirically optimizing parameters is crucial. Photobleaching, as indicated by a decrease in the baseline fluorescence intensity over time, is a harbinger of cell stress and should be avoided.
The visuality of imaging data can lead investigators to favor qualitative, rather than quantitative, analyses. However, as with any modality, reproducible conclusions require quantitation. Quantitative image analysis contains far more complexity than can be covered in a single manuscript, but it is worthwhile to highlight a few key points. First, consistent quantitation requires consistent acquisition. An analysis pipeline developed for a given set of acquisition parameters may not be effective for images acquired at different frequencies, exposure times, laser powers, or wavelengths. Second, the multiplicity of infection has an outsized influence on the frequency of virally induced calcium signals. As such, it is important to evaluate the number of infected cells per field-of-view and ensure consistency across treatment conditions. Alternatively, normalizing signal frequency to the number of infected cells per field-of-view can help reduce error. Finally, while the recently developed mNG-based calcium indicator (NEMO) offers a promising approach for determining absolute Ca2+ concentrations in cells40, most biosensors convey only relative information about biological systems. Thus, background subtraction, normalization, and comparison to a control are critical.
While this protocol focuses on calcium imaging during viral infection, the general approach is readily adaptable. Viral infection can easily be substituted for an alternative treatment condition, and there is an ever-growing litany of biosensors to monitor many different cellular pathways and processes. With any protocol adaptation, investigators must keep analysis top-of-mind, designing experiments with clear, quantifiable outcomes.
Overall, live imaging provides unrivaled temporal resolution, enhancing the characterization of highly dynamic processes. Incorporating organoids into live imaging experiments allows for the observation of these processes in a true-to-tissue, physiologically relevant model. We hope that the protocol described here facilitates experimental design and optimization, empowering researchers to uncover novel aspects of cell and tissue signaling.

Calcium is a widely conserved second messenger that plays a critical role in regulating cellular physiology1. Given its strong charge, small size, and high solubility in physiological conditions, calcium is an ideal manipulator of protein conformation. This makes calcium a powerful means to transduce electrochemical signals into enzymatic, transcriptional, or post-transcriptional alterations. The strict calcium concentration gradients across the endoplasmic reticulum (ER) and plasma membranes create a high driving force that allows for rapid changes in cytosolic calcium concentration. Multiple mechanisms, including both buffering and active transport, tightly maintain this gradient. While necessary for normal cellular functions, this maintenance is energetically expensive, making it particularly susceptible in states of stress 2.
As such, dysregulation of calcium within the cytosol is a near-universal signal of many kinds of cellular stress. Metabolic disturbances, toxins, pathogens, mechanical damage, and genetic perturbations can all disrupt calcium signaling. Regardless of the stimulus, on a whole-cell level, sustained, uncontrolled rises in cytosolic calcium can promote apoptosis and&#160;eventually necrosis3,4. Alterations in cytosolic calcium levels of lower amplitude or higher frequency, however, have varying effects2. Likewise, the outcomes of calcium fluctuations may differ based on the spatial microdomain in which they occur5. Monitoring calcium levels can&#160;therefore offer insight into dynamic signaling processes, but this requires sampling with relatively high temporal and spatial resolution.
Genetically encoded calcium indicators (GECIs) are powerful tools for continuous sampling in live-cell systems6. Some of the most widely used GECIs are GFP-based calcium-responsive fluorescent proteins known as GCaMPs7. The canonical GCaMP is a fusion of three distinct protein domains: a circularly permuted GFP (cpGFP), calmodulin, and M136. The calmodulin domain undergoes a conformation change upon binding calcium, allowing its interaction with M13. The calmodulin-M13 interaction induces a conformational change in the cpGFP that increases its fluorescent emission upon excitation. As such, an increase in calcium concentration correlates with an increase in GCaMP fluorescence intensity. These sensors can be cytosolic or targeted to specific organelles8.
Similar to most tissues, calcium regulates a variety of functions within the gastrointestinal epithelium. The intestinal epithelium is integral for nutrient and fluid absorption but also must form a tight barrier and immune interface to avoid pathogen invasion or toxic insults. Calcium-dependent pathways influence nearly all of these vital functions9,10,11. However, calcium signaling within the intestinal epithelium remains an underexplored frontier with promising potential as a therapeutic target. While monitoring calcium dynamics within the intestinal epithelium in vivo continues to present challenges, human intestinal organoids (HIOs) offer an adaptable ex vivo system for experimentation12. HIOs are 3-dimensional (3D) spheroids derived from human intestinal stem cells and, upon differentiation, recapitulate much of the cellular diversity of the native intestinal epithelium12.
This protocol describes comprehensive methods to engineer HIOs that express GECIs and then prepare engineered HIOs as monolayers for live-cell calcium imaging. It offers viral infection as an example of a pathologic manipulation that disrupts calcium signaling and provides an analytic approach to quantify these changes.

<table><tbody><tr><td>Advanced DMEM F12</td><td>Gibco</td><td>12634028</td><td></td></tr><tr><td>[Leu15]-Gastrin I</td><td>Sigma-Aldrich</td><td>G9145</td><td></td></tr><tr><td>0.05% Trypsin EDTA&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>25300054</td><td></td></tr><tr><td>0.05% Trypsin EDTA&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>25300054</td><td></td></tr><tr><td>1.5mL microcentrifuge tubes</td><td>Fisherbrand</td><td>5408137</td><td></td></tr><tr><td>15mL conical tubes</td><td>Thermofisher Scientific</td><td>0553859A</td><td></td></tr><tr><td>16% formaldehyde</td><td>Thermofisher Scientific</td><td>28906</td><td></td></tr><tr><td>1M HEPES</td><td>Gibco</td><td>15630080</td><td></td></tr><tr><td>1M HEPES</td><td>Gibco</td><td>15630080</td><td></td></tr><tr><td>1X PBS</td><td>Corning&amp;nbsp;</td><td>21-040-CV</td><td></td></tr><tr><td>25 gauge needle</td><td>Thermofisher Scientific</td><td>1482113D</td><td></td></tr><tr><td>A-83-01</td><td>Tocris</td><td>2939</td><td></td></tr><tr><td>ADP</td><td>Sigma-Aldrich&amp;nbsp;</td><td>A2754</td><td></td></tr><tr><td>Advanced DMEM F12</td><td>Gibco</td><td>12634028</td><td></td></tr><tr><td>Antibiotic-antimycocytic&amp;nbsp;</td><td>Gibco</td><td>15240062</td><td></td></tr><tr><td>Antibiotic-antimycotic&amp;nbsp;</td><td>Gibco</td><td>15240062</td><td></td></tr><tr><td>B27 Supplement</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>Bovine serum albumin</td><td>FisherScientific&amp;nbsp;</td><td>BP1600100</td><td></td></tr><tr><td>CellView Cell Culture Slide, PS, 75/25 MM, Glass Bottom, 10 compartments</td><td>Greiner</td><td>543979</td><td></td></tr><tr><td>Collagen IV</td><td>Sigma Aldrich</td><td>C5533</td><td></td></tr><tr><td>DAPI</td><td>Thermofisher Scientific</td><td>D1306</td><td></td></tr><tr><td>EDTA</td><td>Corning</td><td>46-034-CI</td><td></td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>35010CV</td><td></td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>35010CV</td><td></td></tr><tr><td>Fluorobrite</td><td>Gibco</td><td>A1896701</td><td></td></tr><tr><td>GlutaMAX&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>35050079</td><td></td></tr><tr><td>GlutaMAX&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>35050079</td><td></td></tr><tr><td>Human epidermal growth factor</td><td>ProteinTech</td><td>HZ-1326</td><td></td></tr><tr><td>Lentivirus</td><td>VectorBuilder</td><td>(variable)</td><td></td></tr><tr><td>Matrigel</td><td>BD Biosceicen</td><td>356231/CB40230C</td><td></td></tr><tr><td>N2 Supplement</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>N-acetylcysteine</td><td>Sigma-Aldrich</td><td>A9165-5G</td><td></td></tr><tr><td>NH4Cl</td><td>Sigma-Aldrich&amp;nbsp;</td><td>A9434</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma-Aldrich</td><td>N0636</td><td></td></tr><tr><td>Nunc Cell Culture Treated 24-well Plates</td><td>Thermofisher Scientific</td><td>142475</td><td></td></tr><tr><td>Polybrene</td><td>MilliporeSigma</td><td>TR1003G</td><td></td></tr><tr><td>SB202190</td><td>Sigma-Aldrich</td><td>S70767</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher BioReagents</td><td>BP151100</td><td></td></tr><tr><td>TrypLE Express Enzyme, no phenol red</td><td>Thermofisher Scientific</td><td>12604013</td><td></td></tr><tr><td>Trypsin</td><td>Worthington Biochemical</td><td>NC9811754</td><td></td></tr><tr><td>Y-27632</td><td>Tocris</td><td>1254</td><td></td></tr></tbody></table>

live calcium imaging of virus-infected human intestinal organoid monolayers using genetically encoded calcium indicators

Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory activity, actin dynamics, inflammatory responses, stem cell proliferation, and many other uncharacterized cellular functions. As such, mapping calcium signaling dynamics within the intestinal epithelium can provide insight into homeostatic cellular processes and unveil unique responses to various stimuli. Human intestinal organoids (HIOs) are a high-throughput, human-derived model to study the intestinal epithelium and thus represent a useful system to investigate calcium dynamics. This paper describes a protocol to stably transduce HIOs with genetically encoded calcium indicators (GECIs), perform live fluorescence microscopy, and analyze imaging data to meaningfully characterize calcium signals. As a representative example, 3-dimensional HIOs were transduced with lentivirus to stably express GCaMP6s, a green fluorescent protein-based cytosolic GECI. The engineered HIOs were then dispersed into a single-cell suspension and seeded as monolayers. After differentiation, the HIO monolayers were infected with rotavirus and/or treated with drugs known to stimulate a calcium response. An epifluorescence microscope fitted with a temperature-controlled, humidified live-imaging chamber allowed for long-term imaging of infected or drug-treated monolayers. Following imaging, acquired images were analyzed using the freely available analysis software, ImageJ. Overall, this work establishes an adaptable pipeline for characterizing cellular signaling in HIOs.

Figure 1A shows a BMM dome containing 3-dimensional human intestinal organoids that have been transduced to stably express GCaMP6s. Figure 1B shows the same line of organoid replated as a monolayer at 24, 48, and 72 h post-seeding. To validate the function of GCaMP6s, the monolayer was imaged by fluorescence microscopy every 2 s for 4 min, and 100 nM ADP was added to the media after ~20 s. ADP elicits calcium release from the endoplasmic reticulum, increasing cytosolic calcium. The fluorescence intensity detected on the 488 nm channel at each exposure is plotted in Figure 1C. The trace shows a steady baseline fluorescence intensity followed by a rapid increase upon ADP addition, and a gradual return toward baseline. A vehicle control is included to verify that the response is a true signaling event, and not merely a change in fluorescence intensity that may arise from the addition of media.
Scoring an HIO monolayer increases the efficiency of rotavirus infection. While techniques for scoring vary, it is easiest to get consistent infection using a single score along the length of the monolayer, as shown in Figure 2A. When using recombinant strains of rotavirus that express fluorescent proteins, infection can be verified during live imaging (Figure 2B). When using a strain that does not express a marker, infection can be detected by immunofluorescence after imaging. Given the motility of cells in HIO monolayers, it is often difficult to track a single infected cell over time. Instead, use the endpoint staining as a method to verify infection and confirm the multiplicity of infection is matched between fields-of-view (Figure 2C).
Intercellular calcium waves in HIOs can be observed qualitatively, as depicted in Figure 3A. However, determining the frequency of calcium waves in a given field-of-view requires quantitative analysis. The steps of successful segmentation, as described in step 6 of the protocol above, are illustrated in Figure 3A. The figure shows the raw image (Figure 3Ai), the image after the pixel intensities from the previous frame have been subtracted (Figure 3Aii), then the segmented calcium wave (Figure 3Aiii).
Once the calcium waves have been segmented and quantified for each field-of-view over the course of the imaging run, it is often informative to compare the frequency of waves in monolayers exposed to various conditions. The strip chart in Figure 3B shows the total number of calcium waves per field-of-view in mock and rotavirus-inoculated monolayers. This example includes two strains of RV: a strain of human rotavirus (Ito HRV) and a recombinant strain of simian rotavirus SA11 that expresses mRuby (SA11-mRuby). The mock-infected control is taken as the baseline calcium wave frequency, the untreated rotavirus-infected monolayers serve as positive controls, and the rotavirus-infected, treated monolayers are the experimental conditions. Mock-infected and RV-infected monolayers treated with drugs, including the ADP receptor inhibitor, BPTU, can then be compared to both control conditions by one-way ANOVA. The data suggests that BPTU effectively decreases the frequency of intercellular calcium waves.
<img alt="Fluorescence microscopy, cell spheroids comparison, GCaMP6s fluorescence chart, ADP response analysis." class="xfigimg" src="/files/ftp_upload/66132/66132fig01.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1: Validation of GCaMP6s expression in jejunal human intestinal organoids. (A) After lentivirus transduction, the GCaMP-expressing HIOs underwent selection and multiple passages, shown here at 7 days since the most recent passage. Upon stimulation with the 488 nm laser, GCaMP6s expression is evident in the transduced HIOs plated in 15 μL basement membrane matrix as 3-dimensional spheroids (Ai). 50 ms exposures were acquired at 50% laser power using a 20x objective, then stitched together to visualize the entire basement membrane matrix dome. A similar process was performed using 4 ms brightfield exposures (Aii). In the brightfield image, the dark HIOs in the center of the dome are an indication that the cultures are ready for passage. (B) HIOs were enzymatically digested into a single-cell suspension, then re-plated at a density of 150,000 cells per well (5mm in diameter) of an imaging-bottom plate pre-coated with collagen IV. By 72 h post-seeding, the monolayer was confluent and ready for downstream applications. To validate the function of GCaMP, images were acquired using the 488 nm filter set with 50 ms exposures at 50% laser power every 2 s for 4 min. (C) The traces shown here represent the whole-field fluorescence intensity normalized to the first acquisition over the course of imaging. At around 20 s (arrow), after the tenth exposure, 100 nM adenosine diphosphate (ADP) was added to elicit a spike in cytosolic calcium (red). The addition of an equal volume of 1x PBS did not elicit a meaningful response (blue). <a href="https://www.jove.com/files/ftp_upload/66132/66132fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Static equilibrium diagram with needle; fluorescence microscopy; cellular imaging and analysis." class="xfigimg" src="/files/ftp_upload/66132/66132fig02.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2: Scoring an HIO monolayer to facilitate​ RV infection. (A) The figure illustrates a suitable technique for scoring a monolayer using a 25G needle. (B) An HIO monolayer was infected with a recombinant strain of RV that expresses the mRuby fluorescent protein (magenta, arrow), allowing for the identification of infected cells. Alternatively, when using a naturally occurring strain of RV, infected cells can be identified using immunofluorescence. (C) The images shown are from 4 different HIO monolayers after fixation, DAPI staining (blue), and antibody labeling of rotavirus antigen (magenta). Both techniques illustrate the preferential RV infection near the scored portion of the monolayer. <a href="https://www.jove.com/files/ftp_upload/66132/66132fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescence microscopy and bar graph; Ca²⁺ wave analysis in HRV/BPTU study; statistical comparison." class="xfigimg" src="/files/ftp_upload/66132/66132fig03.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3: Segmentation and quantitation of RV-induced intercellular calcium waves. The 488 nm exposures of RV-infected HIOs expressing GCaMP6s were acquired every 1 min for 16 h using the protocol. (A) The figure illustrates the process of intercellular calcium wave segmentation. First, the pixel values from the previous acquisition (t-1) are subtracted from each raw image (Ai) to quantify the change in fluorescence intensity (Aii). A threshold based on an experimentally optimized minimum increase in intensity from the previous frame is then applied. The images are then filtered for a minimum contiguous signal area to select for intercellular calcium waves (Aiii) and exclude single-cell calcium signals. This processing allows for the quantification of calcium waves at each field-of-view. In this experiment, monolayers were either mock-infected, infected with the ITO strain of human rotavirus (HRV), or infected with a recombinant strain of simian rotavirus encoding mRuby on gene segment 7, downstream of non-structural protein 3 (SA11-mRuby). Infected monolayers were either treated with the ADP receptor inhibitor BPTU or left untreated. Monolayers were then imaged at 8 different positions, and calcium waves in each field-of-view were quantified using the pipeline described in this protocol. The points on the strip chart indicate the number of calcium waves detected at each position superimposed on a bar chart marking the mean and SD. One-way ANOVA followed by Dunnett's tests show that while either strain of RV increases the frequency of intercellular calcium waves above that of the uninfected group, BPTU treatment prevents the RV-mediated increase in calcium wave frequency. **p&lt;0.01, ***p&lt;0.001. <a href="https://www.jove.com/files/ftp_upload/66132/66132fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Transduction</td>
			<td>Control</td>
		</tr>
		<tr>
			<td>Y-27632 (1 mM stock) </td>
			<td>4 μL</td>
			<td>4 μL</td>
		</tr>
		<tr>
			<td>Polybrene (1 mg/μL stock)</td>
			<td>2 μL</td>
			<td>2 μL</td>
		</tr>
		<tr>
			<td>Lentivirus</td>
			<td>variable*</td>
			<td></td>
		</tr>
		<tr>
			<td>HighWnt-WRNE *see notes</td>
			<td>to 400 μL</td>
			<td>to 400 μL</td>
		</tr>
	</tbody>
</table>
Table 1: Lentivirus transduction medium. Prepare the transduction media and control media by combining the reagents in the volumes indicated in the second and third columns, respectively. Aim for a multiplicity of infection (MOI) of 10 lentivirus transduction units per cell. For LV packaged in-house, this usually requires ~25-50 μL of concentrated LV. The titer of most commercially packaged LV will be provided with the Certificate of Analysis.
Supplementary Coding File 1: ImageJ macro script to allow for calcium wave quantitation by batch processing. <a href="https://www.jove.com/files/ftp_upload/66132/Wave_counter.zip" target="_blank">Please click here to download this File.</a>

This protocol describes an approach for performing calcium imaging in virus-infected human intestinal organoids and offers an approach to analysis.

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory ...

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-coated one milliliter tip, pipette gently to detach BMM from the plate. Transfer the suspension to a pre-coated 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-coated 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 minus with 10%FBS and gently mix. Centrifuge and add one milliliter of CMGF minus 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 minus. 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 m-Ruby, 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.

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Research

JoVE Journal

Immunology and Infection

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

This video introduces the concepts behind calcium imaging and includes an example protocol for time-lapse imaging of worms contained in molded agarose wells.

Calcium Imaging: A Method to Visualize Neural Activity in Live C. elegans

Source: Fried, D. E., Gulbransen, B. D. In Situ Ca2+ Imaging of the Enteric Nervous System. J. Vis. Exp. (2015)This video demonstrates calcium imaging to visualize and analyze the activity of enteric neurons and glia in mouse Colonic Myenteric plexus preparations.

Calcium Imaging of Enteric Neuron and Glia Activities in a Mouse Colonic Myenteric Plexus

Source: Doser, R., et al. Subcellular Imaging of Neuronal Calcium Handling In Vivo. J. Vis. Exp. (2023)This video demonstrates in vivo imaging of subcellular calcium flux in the ventral nerve cord neurons of transgenic Caenorhabditis elegans strains. Two distinct calcium indicators are employed: a cytoplasmic indicator in one strain and a mitochondrial indicator in another. Neurons are observed under a fluorescence microscope to monitor real-time changes in fluorescence intensity within the...

Imaging Subcellular Calcium Dynamics in Neurons of Caenorhabditis elegans

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

In Situ Ca2+ Imaging of the Enteric Nervous System

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

Here, we present a protocol in which single cells are monitored for acute events and productive HIV-1 infection on a nanofluidic device. Imaging data define virus-host receptor interactions and signaling pathway dynamics. This is the first method for nanofluidic high-throughput longitudinal single-cell culture and imaging to study signaling kinetics and molecular interactions.

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Live-cell imaging of calcium signaling is utilized extensively to study calcium mobilization in stable cell lines grown in monolayers. This protocol describes live-cell fluorescence imaging of calcium signaling using live-cell dyes on complex, primary tissue, and also utilizes machine learning software to quantify the fluorescence intensity of thousands of individual cells simultaneously, thereby streamlining analyses.

Fluorescence-Based Calcium Imaging in Primary Human Airway Epithelial Cultures Using Automated Cell Segmentation

Learn live calcium imaging in virus-infected human intestinal organoids using genetically encoded calcium indicators. This method reveals how rotavirus disrupts epithelial signaling and dysregulates neighboring cells.

Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators

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