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...

Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers

<ol>
	<li>Bootman, M. D., Bultynck, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+cellular+calcium+signaling:+A+primer.">Fundamentals of cellular calcium signaling: A primer.</a> Cold Spring Harb Perspect Biol. 12 (1), a038802 (2020).</li><li>Clapham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling.">Calcium signaling.</a> Cell. 131 (6), 1047-1058 (2007).</li><li>Danese, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+as+a+result+of+calcium+signaling+modulation:+A+cancer-centric+prospective.">Cell death as a result of calcium signaling modulation: A cancer-centric prospective.</a> Biochim Biophys Acta Mol Cell Res. 1868 (8), 119061 (2021).</li><li>Harr, M. W., Distelhorst, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+and+autophagy:+Decoding+calcium+signals+that+mediate+life+or+death.">Apoptosis and autophagy: Decoding calcium signals that mediate life or death.</a> Cold Spring Harb Perspect Biol. 2 (10), a005579 (2010).</li><li>Barak, P., Parekh, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+through+Ca2++microdomains+from+store-operated+CRAC+channels.">Signaling through Ca2+ microdomains from store-operated CRAC channels.</a> Cold Spring Harb Perspect Biol. 12 (7), a035097 (2020).</li><li>Nakai, J., Ohkura, M., Imoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+signal-to-noise+Ca(2+)+probe+composed+of+a+single+green+fluorescent+protein.">A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein.</a> Nat Biotechnol. 19 (2), 137-141 (2001).</li><li>Erofeev, A. I., Vinokurov, E. K., Vlasova, O. L., Bezprozvanny, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GCaMP,+a+family+of+single-fluorophore+genetically+encoded+calcium+indicators.">GCaMP, a family of single-fluorophore genetically encoded calcium indicators.</a> J Evol Biochem Phys. 59 (4), 1195-1214 (2023).</li><li>Suzuki, J., Kanemaru, K., Iino, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+fluorescent+indicators+for+organellar+calcium+imaging.">Genetically encoded fluorescent indicators for organellar calcium imaging.</a> Biophys J. 111 (6), 1119-1131 (2016).</li><li>N&#225;szai, M., Cordero, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+stem+cells:+Got+calcium.">Intestinal stem cells: Got calcium.</a> Curr Biol. 26 (3), R117-R119 (2016).</li><li>Barrett, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-mediated+chloride+secretion+in+the+intestinal+epithelium:+Significance+and+regulation.">Calcium-mediated chloride secretion in the intestinal epithelium: Significance and regulation.</a> Curr Top Membr. 53, 257-282 (2002).</li><li>Xu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-sensing+receptor+regulates+intestinal+dipeptide+absorption+via+Ca2++signaling+and+IKCa+activation.">Calcium-sensing receptor regulates intestinal dipeptide absorption via Ca2+ signaling and IKCa activation.</a> Physiol Rep. 8 (1), e14337 (2020).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+development+and+disease+with+organoids.">Modeling development and disease with organoids.</a> Cell. 165 (7), 1586-1597 (2016).</li><li>Lin, S. C., Haga, K., Zeng, X. L., Estes, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+CRISPR&#8211;Cas9-mediated+genetic+knockout+human+intestinal+tissue&#8211;derived+enteroid+lines+by+lentivirus+transduction+and+single-cell+cloning.">Generation of CRISPR&#8211;Cas9-mediated genetic knockout human intestinal tissue&#8211;derived enteroid lines by lentivirus transduction and single-cell cloning.</a> Nat Protoc. 17 (4), 1004-127 (2022).</li><li>Crawford, S. E., Ramani, S., Blutt, S. E., Estes, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+to+dissect+gastrointestinal+virus-host+interactions:+What+have+we+learned.">Organoids to dissect gastrointestinal virus-host interactions: What have we learned.</a> Viruses. 13 (6), 999 (2021).</li><li>Lambert, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FPbase:+a+community-editable+fluorescent+protein+database.">FPbase: a community-editable fluorescent protein database.</a> Nat Methods. 16 (4), 277-278 (2019).</li><li>Lai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+calcium-triggered+secretion+by+hydrocarbon-stapled+peptides.">Inhibition of calcium-triggered secretion by hydrocarbon-stapled peptides.</a> Nature. 603 (7903), 949-956 (2022).</li><li>Chang-Graham, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rotavirus+induces+intercellular+calcium+waves+through+ADP+signaling.">Rotavirus induces intercellular calcium waves through ADP signaling.</a> Science. 370 (6519), eabc3621 (2020).</li><li>Lee, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anoctamin+1/TMEM16A+controls+intestinal+Cl&#8722;+secretion+induced+by+carbachol+and+cholera+toxin.">Anoctamin 1/TMEM16A controls intestinal Cl&#8722; secretion induced by carbachol and cholera toxin.</a> Exp Mol Med. 51 (8), 1-14 (2019).</li><li>Saha, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+TMEM16A+control+luminal+chloride+secretion+in+a+NHERF1+dependent+manner.">Intestinal TMEM16A control luminal chloride secretion in a NHERF1 dependent manner.</a> Biochem Biophys Rep. 25, 100912 (2021).</li><li>Mroz, M. S., Keely, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+chronically+upregulates+Ca2+-dependent+Cl&#8722;+conductance+and+TMEM16A+expression+in+intestinal+epithelial+cells.">Epidermal growth factor chronically upregulates Ca2+-dependent Cl&#8722; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+role+of+Ca2+-activated+Cl&#8722;+channel+transmembrane+member+16A+in+lipopolysaccharide-induced+intestinal+epithelial+barrier+dysfunction+in+vitro.">Dual role of Ca2+-activated Cl&#8722; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enterochromaffin+cells+are+gut+chemosensors+that+couple+to+sensory+neural+pathways.">Enterochromaffin cells are gut chemosensors that couple to sensory neural pathways.</a> Cell. 170 (1), 185-198.e16 (2017).</li><li>Paradis, T., B&#232;gue, H., Basmaciyan, L., Dalle, F., Bon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+junctions+as+a+key+for+pathogens+invasion+in+intestinal+epithelial+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium/Ask1/MKK7/JNK2/c-Src+signalling+cascade+mediates+disruption+of+intestinal+epithelial+tight+junctions+by+dextran+sulfate+sodium.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+integration+by+Ca2++regulates+intestinal+stem+cell+activity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+host+cell+calcium+signaling+during+viral+infections:+Emerging+paradigm+with+high+clinical+relevance.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rotavirus+calcium+dysregulation+manifests+as+dynamic+calcium+signaling+in+the+cytoplasm+and+endoplasmic+reticulum.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rotavirus+disrupts+calcium+homeostasis+by+NSP4+viroporin+activity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rotavirus+NSP4+viroporin+domain+is+a+calcium-conducting+ion+channel.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+hijacked+through+viroporin-activated+calcium/calmodulin-dependent+kinase+kinase-&#946;+signaling+is+required+for+rotavirus+replication.">Autophagy hijacked through viroporin-activated calcium/calmodulin-dependent kinase kinase-&#946; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COPII+vesicle+transport+is+required+for+Rotavirus+NSP4+interaction+with+the+autophagy+protein+LC3+II+and+trafficking+to+viroplasms.">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., I&#353;a, P., Arias, C. F., L&#243; Pez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+calcium+on+the+early+steps+of+Rotavirus+infection.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiological+consequences+of+calcium-conducting+viroporins.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovirus+NS1-2+has+viroporin+activity+that+induces+aberrant+cellular+calcium+signaling+to+facilitate+virus+replication.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+colonoid+monolayers+to+study+interactions+between+pathogens,+commensals,+and+host+intestinal+epithelium.">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., Ord&#243;&#241;ez-Mor&#225;n, P., Imajo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials+for+intestinal+organoid+technology+and+personalized+disease+modeling.">Biomaterials for intestinal organoid technology and personalized disease modeling.</a> Acta Biomater. 132, 272-287 (2021).</li><li>Cevallos Porta, D., L&#243;pez, S., Arias, C. F., Isa, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarized+rotavirus+entry+and+release+from+differentiated+small+intestinal+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Norovirus+efficiently+replicates+in+differentiated+3D-human+intestinal+enteroids.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototoxicity+in+live+fluorescence+microscopy,+and+how+to+avoid+it.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+NEMO+as+calcium+indicators+with+large+dynamics+and+high+sensitivity.">Engineering of NEMO as calcium indicators with large dynamics and high sensitivity.</a> Nat Methods. 20 (6), 918-924 (2023).</li></ol>

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 osmoti...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</td>
			<td>Gibco&#160;</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% Trypsin EDTA&#160;</td>
			<td>Gibco&#160;</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&#160;</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&#160;</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&#160;</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic&#160;</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&#160;</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&#160;</td>
			<td>Corning&#160;</td>
			<td>35010CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</td>
			<td>Corning&#160;</td>
			<td>35010CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorobrite</td>
			<td>Gibco</td>
			<td>A1896701</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX&#160;</td>
			<td>Gibco&#160;</td>
			<td>35050079</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX&#160;</td>
			<td>Gibco&#160;</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&#160;</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

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.

Author Spotlight: Investigating Viral Disruption of Intestinal Epithelial Signaling &amp;#8211; Research Insights and Future Directions 

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

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 and live virus-infected epithelial tissue. By engineering organoids to express genetically encoded calcium indicators, our approach allows for a 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.

Figure 1A shows a BMM dome containing 3-dimensional human intestinal organoids that have&#160;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, increasi...

Watch this Scientific Journal Video about Investigating Viral Disruption of Intestinal Epithelial Signaling – Research Insights and Future Directions at JoVE.com

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 ...

live-calcium-imaging-virus-infected-human-intestinal-organoid

Research

JoVE Journal

Immunology and Infection

754 Views.  Baylor College of Medicine. Attraverso il nostro lavoro, miriamo a capire come i virus interrompono la segnalazione all'interno dell'epitelio intestinale. Siamo particolarmente interessati a comprendere i modi in cui le cellule infettate da virus disregolano le cellule non infette vicine per contribuire alla patogenesi. Recentemente abbiamo scoperto che le cellule infettate da rotavirus rilasciano impulsi di ADP, che disregola le cellule non infette vicine e facilita la diffusione virale ed esacerba la malattia.S...

Video: Autore in primo piano: Indagine sull'interruzione virale della segnalazione epiteliale intestinale - Approfondimenti di ricerca e direzioni future 