Name: whole cell electrophysiology of primary cultured murine enterochromaffin cells
Uploaded: 2018-09-26T13:30:00
Description: Watch this Scientific Journal Video about Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells at JoVE.com

The authors thank Mrs. Lyndsay Busby for administrative assistance and Mr. Robert Highet from Mayo Clinic Division of Engineering for design and 3D printing of the culture dish insert. This work was supported by NIH K08 to AB (DK106456), Pilot and Feasibility Grant to AB from Mayo Clinic Center for Cell Signaling in Gastroenterology (NIH P30DK084567) and 2015 American Gastroenterological Association Research Scholar Award (AGA RSA) to AB and NIH R01 to GF (DK52766).

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Mayo Clinic. The primary cell culture portion of this protocol is based on previously published methods that can be referenced for further details10. All experimental procedures must be approved by the (IACUC), and all experiments must be performed in accordance with relevant guidelines and regulations.
1. Culture Preparation
<ol>
	<li>Media.
	<...

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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=Sodium+channel+NaV1.3+is+important+for+enterochromaffin+cell+excitability+and+serotonin+release.">Sodium channel NaV1.3 is important for enterochromaffin cell excitability and serotonin release.</a> Scientific Reports. 7 (15650), (2017).</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 (2017).</li><li>Alcaino, C., Knutson, K., Gottlieb, P. A., Farrugia, G., Beyder, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensitive+ion+channel+Piezo2+is+inhibited+by+D-GsMTx4.">Mechanosensitive ion channel Piezo2 is inhibited by D-GsMTx4.</a> Channels. 11 (3), 245-253 (2017).</li><li>Kim, M., Javed, N. H., Yu, J. G., Christofi, F., Cooke, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stimulation+activates+Galphaq+signaling+pathways+and+5-hydroxytryptamine+release+from+human+carcinoid+BON+cells.">Mechanical stimulation activates Galphaq signaling pathways and 5-hydroxytryptamine release from human carcinoid BON cells.</a> Journal of Clinical Investigation. 108 (7), 1051-1059 (2001).</li><li>Modlin, I. M., Kidd, M., Pfragner, R., Eick, G. N., Champaneria, M. 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(122), 55687 (2017).</li><li>Raghupathi, 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=Identification+of+unique+release+kinetics+of+serotonin+from+guinea-pig+and+human+enterochromaffin+cells.">Identification of unique release kinetics of serotonin from guinea-pig and human enterochromaffin cells.</a> The Journal of Physiology. 591, 5959-5975 (2013).</li><li>Nozawa, 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=TRPA1+regulates+gastrointestinal+motility+through+serotonin+release+from+enterochromaffin+cells.">TRPA1 regulates gastrointestinal motility through serotonin release from enterochromaffin cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (9), 3408-3413 (2009).</li><li>Reimann, F., 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=Characterization+and+functional+role+of+voltage+gated+cation+conductances+in+the+glucagon-like+peptide-1+secreting+GLUTag+cell+line.">Characterization and functional role of voltage gated cation conductances in the glucagon-like peptide-1 secreting GLUTag cell line.</a> The Journal of Physiology. 563, 161-175 (2005).</li></ol>

Fully understanding EC cell function requires a high-quality primary culture method to generate cells for whole cell electrophysiology. Primary cultures of GI epithelium had traditionally been difficult due to low survival. Alleviating this confounding factor, the present method yields cultures of singular EC cells from either small or large bowel that are capable of surviving for several days.
We found that the following were critical for improving the quality of cultures to be suitable for e...

The gastrointestinal (GI) epithelium is a diverse community consisting of several cell types. Enteroendocrine cells comprise roughly 1% of all epithelial cells, and enterochromaffin (EC) cells are the largest enteroendocrine cell population1. Recent studies show that enteroendocrine2 and EC3,4 cells are electrically excitable. We are interested in understanding primary EC cell electrophysiology. Thus, the purpose of this study was to establish primary EC cell cultures optimized for whole cell electrophysiology.
Existing EC cell lines that produce and secrete 5-HT (e.g.,&#160;QGP-15, BON6, KRJ-17) and have been used to examine electrophysiology5,8 are typically generated from immortalized neoplastic tissues. While the information acquired from these cell lines is valuable5,8, studies of primary cell electrophysiology are necessary to properly understand EC cell physiology. The electrophysiology of primary EC cells requires the isolation and culture of single epithelial cells, which has been limited by low viability of epithelial cultures.
The culture method presented in this study relies on transgenic mice with fluorescently labeled EC cells, like Tph1-CFP9, used in this study. The method optimizes mixed primary epithelial cultures developed previously2,10 for single cell electrophysiology3. Previous methods used combinations of Trypsin/EDTA plus Collagenase A or EDTA and DTT for enzymatic digestion and used density gradients to specifically isolate and culture guinea-pig11 and rat EC cells12. More recently, intestinal organoids were generated and mechanically disrupted for electrophysiological recordings4. Cultures using these methods are well suited for serotonin release experiments, RT-qPCR analysis and, while they could be used for electrophysiology, are reliant on the success of time consuming organoid generation, density gradients to identify EC cells, and the cellular disruptive effects of Trypsin and EDTA. By contrast, the protocol described here improves enzymatic and mechanical treatments, optimizes culturing conditions, and streamlines procedures to produce single and healthy EC cells suitable for the high cellular standards needed for electrophysiology.
This method will be useful for scientists who want to work on primary EC cells in mixed cultures rather than immortalized cultures and want to investigate the electrophysiology of single cells. However, it may prove less appropriate for studying cell populations that need sorting or long-term cultures that require survival past 72 h.

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			<td height="20" style="height:20px;width:276px;">High Glucose DMEM</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">D6546</td>
			<td style="width:564px;">Store at 4&#730; C for no longer than 1 month</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Fetal Bovine Serum (FBS), Heat Inactivated</td>
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			<td style="width:175px;">F4135</td>
			<td>Freeze in 25 mL aliquots&#160; and store long term at -20&#730; C</td>
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			<td height="20" style="height:20px;width:276px;">L-Glutamine</td>
			<td style="width:179px;">Life Technologies</td>
			<td style="width:175px;">25030-081</td>
			<td>Freeze in 5 mL aliquots and store long term at -20&#730; C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Penicillin/ Streptomycin</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">P0781</td>
			<td>Freeze in 5 mL aliquots and store long term at -20&#730; C</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">A2153</td>
			<td>Store at 4&#730; C for long term storage</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Collagenase XI</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">C9407</td>
			<td>Store at -20&#730; C in aliquots of 100mg, avoid freeze thaw and avoid lot to lot variation</td>
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			<td height="20" style="height:20px;width:276px;">Phosphate Buffered Saline (PBS), w/ Ca and Mg</td>
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			<td style="width:175px;">D8662</td>
			<td>Store long term at&#160;&#160; 4&#730; C</td>
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			<td height="20" style="height:20px;width:276px;">Y-27632 (Rock Inhibitor)</td>
			<td style="width:179px;">StemCell</td>
			<td style="width:175px;">72304</td>
			<td>Resuspend at 5mM and store in small aliquots at -80&#730; C</td>
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			<td height="20" style="height:20px;width:276px;">100x15mm culture dish</td>
			<td style="width:179px;">Corning (Falcon)</td>
			<td>351029</td>
			<td></td>
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			<td height="20" style="height:20px;width:276px;">Transfer Pipette</td>
			<td style="width:179px;">Fisherbrand</td>
			<td style="width:175px;">13-711-7M</td>
			<td></td>
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			<td height="20" style="height:20px;width:276px;">10 mL serological pipette</td>
			<td style="width:179px;">Corning (Falcon)&#160;</td>
			<td style="width:175px;">357551</td>
			<td style="width:564px;"></td>
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			<td height="20" style="height:20px;width:276px;">50 mL Conical</td>
			<td style="width:179px;">Corning (Falcon)&#160;</td>
			<td style="width:175px;">352098</td>
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			<td height="20" style="height:20px;width:276px;">15 mL Conical</td>
			<td style="width:179px;">Corning (Falcon)&#160;</td>
			<td style="width:175px;">352097</td>
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			<td height="20" style="height:20px;width:276px;">1000 mL Barrier Pipet Tips</td>
			<td style="width:179px;">Thermo Scientific (Art)</td>
			<td style="width:175px;">2079E</td>
			<td>Keep sterile</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Matrigel, Growth Factor Reduced</td>
			<td style="width:179px;">Corning</td>
			<td style="width:175px;">354230</td>
			<td>Store at 150uL aliquots at -20&#730; C. Thaw on ice and keep cold until ready for plating</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">MatTek Glass Bottom Dishes</td>
			<td style="width:179px;">MatTek Corporation</td>
			<td style="width:175px;">P35G-1.5-14-C</td>
			<td>Keep sterile</td>
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		<tr height="35">
			<td height="35" style="height:35px;width:276px;">Micro-dissection Forceps - Very Fine, Extra-Long Points</td>
			<td style="width:179px;">Fine Science Tools</td>
			<td>SB12630M</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Surgical Scissors-Sharp</td>
			<td style="width:179px;">Nasco</td>
			<td>14002-14</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Micro-Dissection Scissors</td>
			<td style="width:179px;">Nasco</td>
			<td style="width:175px;">SB46568M</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Monoject Hypo Needle, 21G x 1&#34; A, 100/BX</td>
			<td style="width:179px;">Covidien</td>
			<td style="width:175px;">8881250172</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">&#160;Tg(Tph1-CFP)1Able/J Mice</td>
			<td style="width:179px;">Jackson</td>
			<td>Stock # 028366</td>
			<td>Use male or female mice between the ages of 4-7 weeks</td>
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		<tr height="35">
			<td height="35" style="height:35px;width:276px;">Microfil nonmetallic syringe needles (34 gauge, 67 mm)</td>
			<td style="width:179px;">World Precision Instruments</td>
			<td>MF34G-5</td>
			<td></td>
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			<td height="20" style="height:20px;width:276px;">Parafilm &#34;M&#34; Laboratory Film</td>
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			<td height="20" style="height:20px;width:276px;">R6101</td>
			<td style="width:179px;">Dow Corning</td>
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			<td height="20" style="height:20px;width:276px;">6-mL syringe with regular luer tip</td>
			<td style="width:179px;">Monoject</td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">3-mL syringe with regular luer tip</td>
			<td style="width:179px;">Monoject</td>
			<td></td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:276px;">Electrophysiology Equipment</td>
			<td></td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;width:276px;">Amplifier</td>
			<td style="width:179px;">Molecular Devices</td>
			<td>Axopatch 200B</td>
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			<td height="20" style="height:20px;width:276px;">Data acquisition system (daq)</td>
			<td style="width:179px;">Molecular Devices</td>
			<td>Digidata 1440A</td>
			<td></td>
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			<td height="20" style="height:20px;width:276px;">Signal conditioner</td>
			<td style="width:179px;">Molecular Devices</td>
			<td>CyberAmp 320</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Software</td>
			<td style="width:179px;">Molecular Devices</td>
			<td>pClamp 10.6</td>
			<td></td>
		</tr>
	</tbody>
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whole cell electrophysiology of primary cultured murine enterochromaffin cells

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory cells, EC cells sense luminal stimuli and convert them into serotonin (5-hyroxytryptamine, 5-HT) release events. However, the electrophysiology of these cells is poorly understood because they are difficult to culture and to identify. The method presented in this paper outlines primary EC cell cultures optimized for single cell electrophysiology. This protocol utilizes a transgenic cyan fluorescent protein (CFP) reporter to identify mouse EC cells in mixed primary cultures, advancing the approach to obtaining high-quality recordings of whole cell electrophysiology in voltage- and current-clamp modes.

Cultures: 
Primary murine epithelial cells made from a transgenic Tph1-CFP model attach to dishes after 4 hours and are ready for physiological experiments between 24 to 72 h. When culture conditions had not been optimized, the epithelial cell culture consisted of large clumps, floating cellular debris, damaged membranes, and weak CFP signal in the EC cells (Figure 1A). Cultures that have been optimized for electrophysio...

Watch this Scientific Journal Video about Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells at JoVE.com

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory ...

This method can help answer key questions in enteric neuroscience and gastrointestinal physiology, such as, what are the mechanisms of enterochromaffin cell excitability and response to luminal stimuli, and what are the mechanisms of gastrointestinal hormone release? The main advantage of this technique is it allows us to study in detail enterochromaffin cells using single-cell techniques, like electrophysiology. Demonstrating the procedure will be co-first authors Kaitlyn Knutson and Peter Strege, the outstanding technologists who developed these techniques.To begin this procedure, place a six-milliliter syringe needle filled with ice-cold PBS into the lumen of the extracted intestinal segment. Use the microdissection forceps, clamp and seal the intestines around the syringe needle, and gently flush six milliliters of PBS through the lumen to rinse away any fecal matter. Next, invert the intestinal tissue by gently weaving the microdissection forceps through the lumen, pinching an intestinal wall, and retracting the tissue proximal to the tip of the forceps.Repeat this process until the segment is inside-out with the lumen facing outward. Using microdissection scissors, cut the tissue segments into one-centimeter pieces. Then transfer the tissue segments into a 50-milliliter beaker filled with five milliliters of sterile PBS.Subsequently, mince the tissue pieces to a liquified consistency. Transfer the minced tissue and 15 milliliters of ice-cold PBS to a clean 50-milliliter tube. Triturate two times with the transfer pipette.Wait for the tissue to settle. Afterward, remove 15 milliliter of the PBS supernatant and replace with fresh PBS. Repeat this process three times, until the PBS is clear.For the first digestion, from the water bath, retrieve one of the 50-milliliter tubes containing 10 milliliters of pre-warmed digestion media. Next, add 250 microliters of the PBS collagenase solution in the tube with the minced intestinal pieces. Finally, minced intestinal tissue pieces were added to the digestion media and PBS collagenase solution.Then, place the sample in a water bath for five minutes at 37 degrees Celsius. Slowly titrate the tissue once with a 10-milliliter serological pipette and briefly let the tissue pieces settle. Use a transfer pipette to remove 10 milliliters of the supernatant.For the second digestion, add 250 microliters of the PBS collagenase solution to the tissue and place it in a water bath at 37 degrees Celsius. Afterward, slowly titrate the solution once with a 10-milliliter serological pipette. Then remove 10 milliliters of the supernatant.For the third digestion, add 250 microliters of the PBS collagenase solution in the tube. Place it in a 37-degree Celsius water bath for 10 minutes. Then shake the tube two to three times per minute at a higher intensity than digestions one and two.Subsequently, slowly pipette the tissue up and down with a 10-milliliter serological pipette and allow the tissue pieces to settle for a short time. Transfer 10 milliliters of the supernatant into a new 15-milliliter tube. Add two milliliters of warmed EC cell complete culture media and invert once to mix.Next, spin the sample at 100 g for five minutes at room temperature. After five minutes, remove the supernatant and re-suspend the remaining pellet in 2.5 milliliters of warmed EC cell complete culture media. For the fourth digestion, repeat the digestion procedures, but increase the incubation time by 15 minutes for colon, and remain at 10 minutes for jejunum.To culture the cells, use a transfer pipette to combine the cell suspensions collected from digestions three and four into a new 15-milliliter tube. Remove a 10-microliter aliquot of cells and count with a hemocytometer. Then spin the remaining cell suspension at 100 g for five minutes at room temperature.After five minutes, remove the supernatant and add EC cell complete culture media using a 5-milliliter serological pipette at a density of one million cells per milliliter. Re-suspend the tissue using a transfer pipette. Subsequently, remove the coated glass-bottom culture dishes from the incubator.Using a P1000 pipette, replace the extra cellular matrix from each culture dish with 250 microliters of the final cell suspension. To achieve a whole cell gigaseal, lower the electrode into the bath solution. With the micromanipulator, maneuver the electrode tip in plane with and directly horizontal from the cell.Then expel 0.2 milliliters of air from the syringe. Gently move the electrode horizontally along the X axis to touch the cell. Watch for a dimple to appear on the EC cell, and an increase in membrane resistance on the seal tests.Apply 0.1 to 0.2 milliliters of suction on the syringe and hold the plunger steady until 100 mega-ohms is achieved. Then release the grip from the plunger. Wait for the cell to gigaseal.Then gently disconnect the syringe. To record whole-cell voltage-gated sodium current, apply a quick tap of suction on a syringe. Repeat until the whole cell access is achieved before gently disconnecting the syringe.Next, turn on the whole cell capacitance compensation and adjust the capacitance and series resistance. Turn on the series resistance compensation. With lag set at 60 microseconds, adjust the series resistance compensation and close the seal test.Record an average of five runs of whole-cell voltage-gate sodium current. Quickly take note of two parameters of the voltage-gated sodium current:The voltage at window current, and the peak sodium current density. To record the elicited action potentials, switch the mode from voltage clamp to current clamp.Load the episodic current clamp protocol. Adjust the holding current to zero picoamp here and turn on the external command. Record the elicited action potentials.Note the least amount of current injected to fire off an action potential. To record spontaneous action potentials, turn off the external command. Load a gap-free current clamp protocol.Change the holding current to the amount of current injected in the last sweep in the elicited protocol that did not fire an action potential. Subsequently, turn on the external command. Double check that the predicted holding current brings the membrane potential below the threshold to fire an action potential.Adjust the holding current if necessary and record the spontaneous activity. Cultures that have been optimized for electrophysiology consisted of single cells and small clumps with bright CFP signal. When culture conditions have not been optimized, the epithelial cell culture consisted of large clumps, floating cellular debris, damaged membranes, and weak CFP signal in the EC cells.EC whole cells were obtained at 30 plus or minus 7%of attempts from colon cultures, and 41 plus or minus 3%of attempts from jejunum culture. By whole cell electrophysiology, sodium currents were recorded from 81.3 plus or minus 4.0%of TPH-1 CPF positive cells from jejunum and 64.1 plus or minus 9.2%of them from colon. Of 59 EC cells from jejunum cultures, 19 EC cells in current clamp mode fired spontaneous AP, 29 EC cells fired AP only when elicited by a step protocol in current clamp mode, and 11 EC cells did not fire AP spontaneously or by step protocol.While attempting this procedure, it's important to remember that the optimal digestion protocol for primary epithelial cultures may be variable between uses, so it's important to determine the optimal amount of agitation to obtain single cells. Following this procedure, other methods, like single cell PCR and fluorescence, can be performed to answer additional questions, like what enterochromaffin cells express and how stimuli are coupled to intercellular signaling. After this development, this technique paved the way for the researches in gastrointestinal physiology to explore how enterochromaffin cells work and how they regulate gastrointestinal motility and secretion in mice.

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A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...