This study was supported by ERC StG TroyCAN (851241). E.E. is a Cancerfonden Postdoctoral Associate. M.G. is a Ragnar S&#246;derberg Fellow and Cancerfonden Junior Investigator. We are grateful for the technical assistance from Karolinska Institutet core facilities, including the Biomedicum Flow Cytometry Core Facility, the Biomedicum Imaging Core (BIC), and the Comparative Medicine Biomedicum (KMB) animal facility. We thank members of the Genander lab for carefully reading and commenting on the protocol.

<ol>
	<li>Sachs, N., 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=Long-term+expanding+human+airway+organoids+for+disease+modeling.">Long-term expanding human airway organoids for disease modeling.</a> The EMBO Journal. 38 (4), 100300 (2019).</li><li>Lohmussaar, K., 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=Patient-derived+organoids+model+cervical+tissue+dynamics+and+viral+oncogenesis+in+cervical+cancer.">Patient-derived organoids model cervical tissue dynamics and viral oncogenesis in cervical cancer.</a> Cell Stem Cell. 28 (8), 1380-1396 (2021).</li><li>Sato, 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=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Smukler, S. R., 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=The+adult+mouse+and+human+pancreas+contain+rare+multipotent+stem+cells+that+express+insulin.">The adult mouse and human pancreas contain rare multipotent stem cells that express insulin.</a> Cell Stem Cell. 8 (3), 281-293 (2011).</li><li>Zheng, 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=A+new+murine+esophageal+organoid+culture+method+and+organoid-based+model+of+esophageal+squamous+cell+neoplasia.">A new murine esophageal organoid culture method and organoid-based model of esophageal squamous cell neoplasia.</a> iScience. 24 (12), 103440 (2021).</li><li>DeWard, A. D., Cramer, J., Lagasse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+heterogeneity+in+the+mouse+esophagus+implicates+the+presence+of+a+nonquiescent+epithelial+stem+cell+population.">Cellular heterogeneity in the mouse esophagus implicates the presence of a nonquiescent epithelial stem cell population.</a> Cell Reports. 9 (2), 701-711 (2014).</li><li>Kasagi, 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=The+esophageal+organoid+system+reveals+functional+interplay+between+notch+and+cytokines+in+reactive+epithelial+changes.">The esophageal organoid system reveals functional interplay between notch and cytokines in reactive epithelial changes.</a> Cellular and Molecular Gastroenterology and Hepatology. 5 (3), 333-352 (2018).</li><li>Plikus, M. V., 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=Fibroblasts:+Origins,+definitions,+and+functions+in+health+and+disease.">Fibroblasts: Origins, definitions, and functions in health and disease.</a> Cell. 184 (15), 3852-3872 (2021).</li><li>McCarthy, N., 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=Distinct+mesenchymal+cell+populations+generate+the+essential+intestinal+BMP+signaling+gradient.">Distinct mesenchymal cell populations generate the essential intestinal BMP signaling gradient.</a> Cell Stem Cell. 26 (3), 391-402 (2020).</li><li>Cordero-Espinoza, 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=Dynamic+cell+contacts+between+periportal+mesenchyme+and+ductal+epithelium+act+as+a+rheostat+for+liver+cell+proliferation.">Dynamic cell contacts between periportal mesenchyme and ductal epithelium act as a rheostat for liver cell proliferation.</a> Cell Stem Cell. 28 (11), 1907-1921 (2021).</li><li>Pastula, 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=Three-dimensional+gastrointestinal+organoid+culture+in+combination+with+nerves+or+fibroblasts:+a+method+to+characterize+the+gastrointestinal+stem+cell+niche.">Three-dimensional gastrointestinal organoid culture in combination with nerves or fibroblasts: a method to characterize the gastrointestinal stem cell niche.</a> Stem Cells International. 2016, 3710836 (2016).</li><li>Hamilton, T. G., Klinghoffer, R. A., Corrin, P. D., Soriano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+divergence+of+platelet-derived+growth+factor+alpha+receptor+signaling+mechanisms.">Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms.</a> Molecular and Cellular Biology. 23 (11), 4013-4025 (2003).</li><li>McGinn, 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=A+biomechanical+switch+regulates+the+transition+towards+homeostasis+in+oesophageal+epithelium.">A biomechanical switch regulates the transition towards homeostasis in oesophageal epithelium.</a> Nature Cell Biology. 23 (5), 511-525 (2021).</li><li>Zhang, Y., Bailey, D., Yang, P., Kim, E., Que, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+stem+cells+of+the+esophagus.">The development and stem cells of the esophagus.</a> Development. 148 (6), (2021).</li><li>Ohlund, D., 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=Distinct+populations+of+inflammatory+fibroblasts+and+myofibroblasts+in+pancreatic+cancer.">Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer.</a> The Journal of Experimental Medicine. 214 (3), 579-596 (2017).</li><li>Pentinmikko, N., 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=Notum+produced+by+Paneth+cells+attenuates+regeneration+of+aged+intestinal+epithelium.">Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium.</a> Nature. 571 (7765), 398-402 (2019).</li><li>Janson, D., Rietveld, M., Willemze, R., El Ghalbzouri, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+serially+passaged+fibroblasts+on+dermal+and+epidermal+morphogenesis+in+human+skin+equivalents.">Effects of serially passaged fibroblasts on dermal and epidermal morphogenesis in human skin equivalents.</a> Biogerontology. 14 (2), 131-140 (2013).</li><li>Landry, N. M., Rattan, S. G., Dixon, I. M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+of+maintaining+primary+murine+cardiac+fibroblasts+in+two-dimensional+cell+culture.">An improved method of maintaining primary murine cardiac fibroblasts in two-dimensional cell culture.</a> Scientific Reports. 9 (1), 12889 (2019).</li></ol>

The authors declare no conflict of interest.

The protocol presented here establishes an in vitro model for investigating functional esophageal epithelial-fibroblast interactions.
The epithelial layer is separated from the stroma, allowing for an optimized dissociation protocol for both the epithelial and stromal compartment. Despite optimization of the epithelial dissociation protocol, tissue clumps remain apparent. Pipetting up and down vigorously every 15 min decreases the number and size of clumps substantially. Other protocols use trypsin to further dissociate the epithelial layer5,6. Here, the use of trypsin, or increasing the dissociation time further, is not recommend, as this tends to result in decreased epithelial cell viability and organoid forming efficiency. In contrast to the epithelium, the stroma is easily dissociated, and 30 min in dissociation solution results in a single-cell suspension with ~90% fibroblast viability (Figure 1E). Excluding the epithelial-stomal separation step in the protocol increases the dissociation time substantially, resulting in a decreased fibroblast viability and a lower yield of epithelial cells. Additionally, separating the epithelium from the stroma provides an opportunity to determine cell numbers of each population and mix epithelial cells and fibroblasts from different mouse lines when setting up the co-cultures.
Studying fibroblast function on organoid growth is a commonly used method in stem cell biology 9,10,11,15,16. Established co-culture media are either DMEM supplemented with 10% fetal calf serum (FCS)9,15 or growth factor reduced medium10,16. In this protocol, the growth factor reduced medium is used to mimic the conditions in the in vivo stem cell niche, where fibroblasts are largely quiescent. FCS is a growth factor rich serum which results in activation and proliferation of fibroblasts in the co-cultures, likely corresponding to a fibroblast cell state distinct from the in vivo state. By excluding FCS and reducing growth factors, so that the medium alone (ERlow) does not support organoid growth and does not stimulate fibroblast proliferation, it is possible to isolate the effect of the fibroblasts on the organoid growth. In this medium, NOGGIN is removed and RSPO reduced to a minimum (10% RPSO). Both NOGGIN and RSPO have been demonstrated to be essential for esophageal organoid growth6. EGF was retained in the co-culture medium, as it does not support organoid growth by itself. However, fibroblasts are also capable of supporting organoid growth in an EGF-reduced medium (ElowRlow; Figure 2B,D).
Organoid co-cultures cannot be sustained through passaging as fibroblasts are lost during trypsinization. However, organoid passaging was included in the protocol since esophageal organoids can be maintained, expanded, and used for further experiment as mono-cultures. Passaged organoids from mono-cultures can be used to set up co-cultures with freshly isolated fibroblasts. A disadvantage of using primary cells is the number of mice needed to set up multiple organoid co-cultures. When focusing on small subpopulations of fibroblasts, the number of co-cultures obtained is limited. In other protocols, fibroblasts are first expanded in culture before using them to set up organoid co-cultures10. However, fibroblasts change morphology and identity during passaging, shown by using primary skin and cardiac fibroblasts17,18. Conventional 2D passaging of esophageal fibroblasts results in both morphology and phenotype changes, demonstrating that in vitro enrichment of fibroblasts is not suitable for co-cultures aiming to phenocopy the endogenous stem cell niche.
Whole mount staining provides a tool to maintain and visualize fibroblast-organoid interaction. It should be noted that, while not all organoids will have fibroblasts directly attached to them, most organoids are in contact with fibroblasts (see Figure 2C). To maintain epithelial-fibroblast interactions, it is important to handle the organoids with care and avoid vigorous pipetting, vortexing, and high-speed spinning. Optimal fixation is important to maintain 3D tissue architecture, as well as keep endogenous fluorescence. A 30 min fixation suffices to retain the H2BeGFP signal and is optimal for the antibodies used in this protocol, however this might vary between the fluorophores and antibodies used. Clearing of the organoids reduces light scattering and improves visualization of the whole 3D structure substantially. As the organoids are small, clearing is easy and fast; however, imaging whole organoids using laser-scanning confocal microscopy can be time-consuming, as multiple Z-stacks need to be made. Confocal microscopes, like the spinning disc, can be used to reduce imaging time.
Overall, esophageal organoids grown in the presence of fibroblasts provide a valuable tool to understand aspects of the esophageal stem cell niche. In addition, whole mount clearing provides an accessible method to visualize the interaction between fibroblasts and organoids.

Organoids are used as 3D in vitro assays to characterize stem and progenitor cells, as well as to understand the signaling cues derived from the cellular components of the stem cell niche1,2,3,4. Mouse esophageal organoids were first described in 2014 and several papers have identified growth factors, like R-Spondin (RSPO), NOGGIN, and epidermal growth factor (EGF), needed to maintain and passage esophageal organoids5,6,7, arguing that similar signaling cues are required for in vivo progenitor cell renewal. However, growth factors are commonly added in non-physiological concentrations, resulting in organoid growth conditions which do not necessarily reflect the in vivo signaling environment.
Fibroblasts are heterogeneous stromal cell populations that support progenitor cell properties in many stem cell niches8. Combining epithelial progenitor cells and fibroblasts in the same organoid culture enables organoid formation in reduced concentrations of exogenously supplemented growth factors. Organoid co-culture systems from intestinal and hepatic epithelia are&#160;described, but a protocol to establish esophageal organoid-fibroblast co-cultures is still outstanding9,10,11.
In this protocol, two fluorescence-activated cell sorting (FACS) strategies for fibroblasts from the esophagus, using either transgenic Pdgfr&#945;H2BeGFP mice12 or wild-type mice with classical antibody staining are outlined. Different subpopulations of fibroblasts can be isolated using cell surface markers of choice, thereby providing flexibility to the protocol. In addition, a 3D fluorescence imaging technique preserving organoid morphology is used to characterize fibroblast-organoid interactions. Organoid clearing provides a quick method to increase the light penetration depth in the organoids, improving the visualization of organoid-fibroblast connections and enabling the recapitulation of the organoid structure in its entirety. This protocol combines esophageal organoid co-culture with a whole mount imaging strategy, enabling functional characterization of the fibroblast-organoid interaction.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>17504001</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#160;Matrigel&#160;Growth Factor Reduced (GFR) Basement Membrane Matrix</td>
			<td>fisher scientific</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>276855-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>11320074</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>14175-129</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Jackson Immuno</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100 solution</td>
			<td>Merck</td>
			<td>93443-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals, Peptides, and recombinant proteins</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI Solution</td>
			<td>Thermo Fisher</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissociation solution: 0.25 mg/ml Liberase TM, 0.25 mg/ml Dnase in HBSS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Sigma-Aldrich</td>
			<td>11284932001</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde, 37%, with 10-15% methanol</td>
			<td>Sigma-Aldrich</td>
			<td>252549-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase</td>
			<td>Sigma-Aldrich</td>
			<td>5401127001</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetyl-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Noggin murine</td>
			<td>Peprotech</td>
			<td>250-38</td>
			<td></td>
		</tr>
		<tr>
			<td>RapiClear 1.47</td>
			<td>SunJin Lab</td>
			<td>#RC147001</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse EGF Protein, CF</td>
			<td>R&#38;D systems</td>
			<td>2028-EG-200</td>
			<td></td>
		</tr>
		<tr>
			<td>R-spondin-1 murine</td>
			<td>Peprotech</td>
			<td>315-32</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX Blue Dead Cell Stain</td>
			<td>Thermo Fisher</td>
			<td>S34857</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermolysin</td>
			<td>Sigma-Aldrich</td>
			<td>T7902-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>Y0503-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic &#38; Glassware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Sterile Cell Strainers 40um</td>
			<td>VWR</td>
			<td>15360801</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Sterile Cell Strainers 70um</td>
			<td>VWR</td>
			<td>431751</td>
			<td></td>
		</tr>
		<tr>
			<td>Menzel Deckgl&#228;ser/ cover slips</td>
			<td>Thermo Fisher</td>
			<td>Q10143263NR15</td>
			<td></td>
		</tr>
		<tr>
			<td>SafeSeal reaction tube, 1.5 mL, PP</td>
			<td>Sarstedt</td>
			<td>72.706</td>
			<td></td>
		</tr>
		<tr>
			<td>Snap Cap Low Retention Microcentrifuge Tubes 0.6 mL</td>
			<td>Thermo Fisher</td>
			<td>3446</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFrost Slides</td>
			<td>VWR</td>
			<td>631-9483</td>
			<td></td>
		</tr>
		<tr>
			<td>Tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.05 mm 4 circular well iSpacer</td>
			<td>SunJin Lab</td>
			<td>#IS204</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 forceps, biology tip</td>
			<td>F.S.T</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmEdge Pen</td>
			<td>VectorLaboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissors Angled to Side Ball Tip 8mm Cutting Edge</td>
			<td>F.S.T</td>
			<td>15033-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope Stellaris 5&#160;</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope ZEISS Stemi 305</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACS ARIA III</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Conjugated Antibodies for FACS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 anti-mouse CD104 Antibody 
			Clone: 346-11A</td>
			<td>123608</td>
			<td>123608</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD26 (DPP-4) Antibody</td>
			<td>H194-112</td>
			<td>H194-112</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cy7 anti-mouse/human CD324 (E-Cadherin) Antibody</td>
			<td>147310</td>
			<td>147310</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies for Immunofluorescence</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD104 (&#223;-integrin 4) 
			Clone: 346-11A</td>
			<td>BioLegend</td>
			<td>553745</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytokeratin 14</td>
			<td>Acris Antibodies (AbD Serotec)</td>
			<td>BP5009</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytokeratin13 
			Clone: EPR3671</td>
			<td>abcam</td>
			<td>ab92551</td>
			<td></td>
		</tr>
		<tr>
			<td>E-cadherin (CD324) 
			Clone: 2.40E+11</td>
			<td>Cell Signaling Technology</td>
			<td>3195</td>
			<td></td>
		</tr>
		<tr>
			<td>Keratin 5 Polyclonal Chicken Antibody, Purified 
			Clone: Poly9059</td>
			<td>BioLegend</td>
			<td>905901</td>
			<td></td>
		</tr>
		<tr>
			<td>p63 
			Clone: 4a4</td>
			<td>abcam</td>
			<td>ab735</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Anti-KLF4 antibod 
			Clone: EPR20753-25</td>
			<td>abcam</td>
			<td>ab214666</td>
			<td></td>
		</tr>
		<tr>
			<td>Vimentin</td>
			<td>Sigma-Aldrich</td>
			<td>AB5733</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-species* antibodies with fluorophore of choice</td>
			<td>Jackson Immuno</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

functional characterization and visualization of esophageal fibroblasts using organoid co-cultures

Epithelial stem and progenitor cells contribute to the formation and maintenance of the epithelial barrier throughout life. Most stem and progenitor cell populations are tucked away in anatomically distinct locations, enabling exclusive interactions with niche signals that maintain stemness. While the development of epithelial organoid cultures provides a powerful tool for understanding the role of stem and progenitor cells in homeostasis and disease, the interaction within the niche environment is largely absent, thereby hindering the identification of factors influencing stem cell behavior. Fibroblasts play a key role in directing epithelial stem and progenitor fate. Here, a comprehensive organoid-fibroblast co-culture protocol enabling the delineation of fibroblast subpopulations in esophageal progenitor cell renewal and differentiation is presented. In this protocol, a method to isolate both epithelial cells and fibroblasts in parallel from the esophagus is described. Distinct fluorescence-activated cell sorting strategies to isolate both the esophageal progenitor cells as well as the fibroblast subpopulations from either transgenic reporter or wild-type mice are outlined. This protocol provides a versatile approach that can be adapted to accommodate the isolation of specific fibroblast subpopulations. Establishing and passaging esophageal epithelial organoid mono-cultures is included in this protocol, enabling a direct comparison with the co-culture system. In addition, a 3D clearing approach allowing for detailed image analysis of epithelial-fibroblast interactions is described. Collectively, this protocol describes a comparative and relatively high-throughput method for identifying and understanding esophageal stem cell niche components in vitro.

The esophagus is divided into different layers: epithelium, lamina propria, submucosa, and muscularis externa (Figure 1A). Fibroblasts reside within the submucosa and lamina propria, referred to as the stroma. In this protocol, the muscularis externa is mechanically removed (Figure 1B), which does not lead to a loss of fibroblasts (Pdgfr&#945;H2BeGFP+) residing in the stroma (Figure 1C). Before dissociation, the epithelium is separated from the stroma resulting in two tissue segments (Figure 1B). Separating the two layers provides the opportunity to increase dissociation time for the more robust epithelial layer compared to the fragile stromal layer. In this way, an efficient isolation protocol yielding both viable epithelial progenitor cells as well as stromal fibroblasts is established (Figure 1B). Esophageal progenitor cells are sorted based on their high INTEGRIN-&#946;4 and E-CADHERIN expression (Figure 1C,D).
Subpopulations of fibroblasts can be isolated by using distinct markers. In this protocol, a strategy for fibroblast isolation based on commonly used fibroblast markers PDGFR&#945;&#160;and DPP4 (CD26) is provided. Isolation by either the Pdgfr&#945;H2BeGFP reporter expression or DPP4 antibody shows that around 50% of the isolated cells are fibroblasts (Figure 1E,F). Additionally, 70% of the PDGFR&#945;+ fibroblasts are DPP4+, indicating that a largely overlapping, but not identical, fibroblast population is obtained. After isolating both epithelial and stromal cell populations, esophageal progenitor cells are either cultured alone or together with fibroblasts in a matrix dome. To study the contribution of fibroblasts to organoid formation, the co-culture is maintained in a growth factor reduced medium (Figure 1G).
Esophageal progenitor cells form organoids in the presence of EGF, NOGGIN and RSPO (ENR). Removing NOGGIN and reducing the amount of RSPO (25 ng/&#181;L; ERlow) is sufficient to prevent organoid formation (Figure 2A). Interestingly, adding either DPP4+ or PDGFR&#945;+ fibroblasts to the esophageal progenitor cells in the ERlow medium restores the organoid forming ability, demonstrating a supportive function for both fibroblast populations (Figure 2A-D). Visualization of the Pdgfr&#945;H2BeGFP transgene shows that fibroblasts are in close contact with the epithelial progenitor cells during organoid formation (Figure 2A). At day 6, Pdgfr&#945;H2BeGFP+&#160;fibroblasts are still abundantly present in the co-culture. Fibroblasts are present throughout the dome, near and touching the organoids (full arrow), or attached to the organoids (arrowhead; Figure 2D).
Whole mount staining shows a 3D representation of the interaction of the fibroblasts with the organoids (Figure 3). While it is possible to perform the whole mount protocol without the use of a clearing solution, it decreases transparency and laser penetration of the organoid (Figure 3B, z-view). When mounting organoids, the spacer helps to maintain organoid morphology. In contrast, plating the coverslip directly on the organoids (without a spacer) flattens the organoids and results in loss of organoid structure (Figure 3A,B).
Both the DPP4+ and PDGFR&#945;+ fibroblasts are found to be wrapped around the organoids (Figure 3C, Video1,&#160;and Video 2). Differentiation of esophageal organoids can be assessed using different markers. Figure 4 shows that the staining protocol provided is suitable for easier-to-stain keratins (KRT14/13) as well as more-difficult-to-stain transcription factors (TRP63/KLF4). The co-culture protocol generates organoids with a similar differentiation pattern, as demonstrated in vivo13,14 and as seen in organoids grown in ENR medium; KRT14+ or TRP63+ progenitor cells form the outer layer and KRT13+ or KLF4+ differentiated cells oriente inwards.
This protocol provides a tool to study the esophageal stem cell niche in vitro and visualizes the interaction between organoids and fibroblasts. By implementing a protocol for the isolation of fibroblasts using antibodies, the method is adaptable and can be used to study fibroblast subpopulations without the need of transgenic mice.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64905/64905fig01.jpg" /> 
Figure 1: Isolation of progenitor cells and fibroblast subpopulations from the esophagus. (A) Schematic overview of the different layers in the esophagus. The stroma contains the lamina propria and submucosa. (B) Schematic overview of the isolation protocol. The muscle (muscularis externa) is mechanically removed using forceps; the remaining esophagus is cut open and incubated in thermolysin to separate the epithelial layer from the stroma. The epithelium and stroma are separated, mechanically minced, and enzymatically digested to single cell suspensions. Dissociated cells are then stained and prepared for FACS. (C) Cross section of the esophagus stripped from the muscularis externa showing Pdgfr&#945;H2BeGFP+ fibroblasts in the stroma. INTEGRIN-&#946;4 (ITG&#946;4) and E-CADHERIN (ECAD) double positive cells are the epithelial progenitor cells of the esophagus. Scale bar = 100 &#181;m. (D) Representative flow cytometry plot of epithelial cell isolation showing the percentage of live cells (upper panel) from all single cells. The lower panel shows the percentage of isolated ITG&#946;4+ ECAD+ progenitor (Prog.) cells from all live cells. (E) Representative flow cytometry plot of stromal cell isolation showing the percentage of live cells (upper left panel). Representative flow cytometry plots showing the percentage of isolated DPP4+ fibroblasts (Fibr.; upper right panel) and Pdgfr&#945;+ fibroblasts (lower left panel) of all live cells. 70% of the Pdgfr&#945;+ fibroblasts are also DPP4+ (lower right panel). (F) Representative flow cytometry plot of the stroma showing DPP4+ only cells (2.5%), DPP4+ PDGFR&#945;+ cells (37.5%), and PDGFR&#945;+ only cells (17.7%). The percentages are of all live cells. (G) Epithelial only cells are plated in a matrix dome in the presence of 50 ng/&#181;L EGF, 100 ng/&#181;L NOGGIN, and 250 ng/&#181;L RSPO (ENR), or together with fibroblasts in the presence of EGF and a low concentration of RSPO (25 ng/&#181;L). <a href="https://www.jove.com/files/ftp_upload/64905/64905fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64905/64905fig02.jpg" /> 
Figure 2: Representative results of organoid co-cultures. (A) Brightfield images showing growth of the organoids from day 1 to day 6. The brightfield images with the organoids co-cultured with Pdgfr&#945;H2BeGFP+ fibroblasts also show the nuclear eGFP signal. Scale bar = 25 &#181;m. (B) Brightfield images of the whole matrix dome at day 6. The left column shows organoid co-cultures grown in the presence of Pdgfr&#945;+ fibroblasts in ERlow or ElowRlow medium. The middle column shows organoid co-cultures grown in the presence of DPP4+ fibroblasts in ERlow or ElowRlow medium. The right column shows organoid mono-cultures grown in ENR medium. ENR medium = EGF (50 ng/&#181;L), NOGGIN (100 ng/&#181;L), and RSPO (250 ng/&#181;L). ERlow = EGF and 25 ng/&#181;L RSPO. ElowRlow = 5 ng/&#181;L EGF and 25 ng/&#181;L RSPO. Scale bar = 500 &#181;m. (C) Graph showing the organoid forming efficiency (%) (i.e., the percentage of cells forming organoids in different culture conditions). Each dot represents a matrix dome and the bar represents the mean of all dots per condition. (D) Brightfield and fluorescent image of day 6 organoids co-cultured with Pdgfr&#945;H2BeGFP+ fibroblasts. Pdgfr&#945;H2BeGFP+ fibroblasts are present throughout the dome, attached to the organoids (arrowhead), and unattached but in contact with the organoids (full arrow). Scale bar = 250 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64905/64905fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64905/64905fig03.jpg" /> 
Figure 3: Whole mount staining protocol for the study of fibroblast-organoid interactions. (A) Schematic overview of the whole mount immunofluorescence protocol. AB = antibody. (B) Immunofluorescence picture of uncleared whole mount staining showing a decreased transparency and penetration of the laser light compared to the cleared organoids. The absence of a spacer results in flattening of the organoid and loss in organoid morphology. (C) Whole mount images of the co-cultured organoids show 3D surfaces of the organoids with VIMENTIN+ fibroblasts (Fibr.) wrapped around and in close contact with the organoid. 3D cross sections and 2D plane images show the lumen of the organoid. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64905/64905fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64905/64905fig04.jpg" /> 
Figure 4: Whole mount images reveal distinct basal and suprabasal cell populations. (A) Whole mount staining of mono- and co-cultured organoids with Pdgfr&#945;H2BeGFP+ fibroblasts showing KRT14+ basal cells in the outer layer and KRT13+ differentiated suprabasal cells. Scale bar = 50 &#956;m. (B) Whole mount staining of mono- and co-cultured organoids with Pdgfr&#945;H2BeGFP+ fibroblasts showing TRP63+ basal cells in the outer layer and KLF4+ differentiated suprabasal cells. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64905/64905fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Table describing the organoid culture media components. <a href="https://www.jove.com/files/ftp_upload/64905/Table1_revised_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Video 1: Pdgfr&#945;H2BeGFP+ fibroblast wrapped around and in close contact with the organoid. The video accompanies the upper panel of Figure 3C. The scale bar&#160;in Figure 3C is 50 &#181;m, and the organoid is ~120 &#181;m in diameter. VIMENTIN is shown in white, E-CADHERIN in red, Pdgfr&#945;H2BeGFP&#160;in green, and DAPI in blue. <a href="https://www.jove.com/files/ftp_upload/64905/Video 1_Re.avi" target="_blank">Please click here to download this Video.</a>
Video 2: DPP4+ fibroblast wrapped around and in close contact with the organoid. The video accompanies the lower panel of Figure 3C. The scale bar&#160;in Figure 3C is 50 &#181;m, and the organoid is ~120 &#181;m in diameter. VIMENTIN is shown in white, E-CADHERIN in red, and DAPI in blue. <a href="https://www.jove.com/files/ftp_upload/64905/Video 2_RE.avi" target="_blank">Please click here to download this Video.</a>

Watch this Scientific Journal Video about Functional Characterization and Visualization of Esophageal Fibroblasts Using Organoid Co-Cultures at JoVE.com

Functional Characterization and Visualization of Esophageal Fibroblasts Using Organoid Co-Cultures

Organoid-fibroblast co-cultures provide a model to study the in vivo stem cell niche. Here, a protocol for esophageal organoid-fibroblast co-cultures is described. Additionally, whole mount imaging is used to visualize the fibroblast-organoid interaction.

Epithelial stem and progenitor cells contribute to the formation and maintenance of the epithelial barrier throughout life. Most stem and progenitor cell ...

functional-characterization-visualization-esophageal-fibroblasts

Research

JoVE Journal

Biology

2.4K Views.  Karolinska Institutet. Organoid-fibroblast co-cultures provide a model to study the in vivo stem cell niche. Here, a protocol for esophageal organoid-fibroblast co-cultures is described. Additionally, whole mount imaging is used to visualize the fibroblast-organoid interaction.

Video: Functional Characterization and Visualization of Esophageal Fibroblasts Using Organoid Co-Cultures

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do not allow for accurate interpretation of this interplay. Using a quantitative imaging-based approach, we present a high-content protocol for characterizing the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations to changes in environmental stimuli. We highlight our ability to distinguish between cell types based upon either fluorescence intensity or inherent morphology features depending on the application. This platform allows for a more comprehensive characterization of subpopulation response to perturbation while utilizing shorter time, smaller amounts of reagents, and lower likelihood of error than traditional cell biology assays. However, in some cases, cell populations may be difficult to identify and quantitate based on complex cellular features and will require additional troubleshooting; we highlight some of these circumstances in the protocol. We demonstrate this application using response to drug in a cancer model; however, it can easily be applied more broadly to other physiological processes. This protocol allows one to identify subpopulations within a co-culture system and characterize the particular response of each to external stimuli.

We have developed a novel protocol for studying heterocellular population dynamics in response to perturbations. This manuscript describes an imaging-based platform that produces quantitative datasets for simultaneous characterization of multiple cellular phenotypes of heterocellular populations in a robust manner.

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...