The authors would like to thank Christopher Fladd and SPARC BioCentre at the Hospital for Sick Children for their help in conducting the membrane potential assays measuring CFTR and ENaC. This work was supported by the CFIT Program with funding provided by CF Canada and the Sick Kids Foundation. This work was funded by the Government of Canada through Genome Canada and the Ontario Genomics Institute (OGI-148). This study was funded by the Government of Ontario. S.X. was supported by the Canadian Association of Gastroenterology (CAG) Ph.D. Scholarship.

1. Cell plating and differentiation
<ol>
	<li>Culture Calu-3 and Caco-2 cells in EMEM containing 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin (pen/strep) in a T-75 flask.</li>
	<li>Once the cells reach 80-100% confluency, aspirate the medium from the T-75 flask.</li>
	<li>Gently wash the cells once with 10 mL of phosphate-buffered saline (PBS).</li>
	<li>Add 2 mL of prewarmed 0.25% Trypsin/0.1% EDTA to the cell monolayer and place the T-75 flask at 37 &#176;C for approximately 5 min to allow the cells to lift from the culturing flask surface and dissociate into a single-cell suspension.</li>
	<li>Once the cells are lifted from the flask surface, neutralize the reaction with 8 mL of culture medium (step 1.1) for a total of 10 mL of cell suspension. 
	NOTE: Mechanically dissociate cell clumps into a single-cell suspension with a 10 mL serological pipette.</li>
	<li>Plate the cells onto a 96-well plate (~140,000-150,000 cells/well) or a 384-well plate (~45,000-50,000 cells/well) at 30-40% confluency.
	<ol>
		<li>To plate one full 96-well plate, add 1.5 mL of the cell suspension to 18.5 mL of the culture medium in a 50 mL conical tube. After mixing, add 200 &#181;L of the cell suspension to each well.</li>
		<li>To plate one full 384-well plate, add 1 mL of the cell suspension to 17 mL of the culture medium in a 50 mL conical tube. After mixing, add 50 &#181;L of the cell suspension to each well. 
		NOTE: Ensure there are no clumps of cells, and single cells are distributed evenly across each well.</li>
	</ol>
	</li>
	<li>Change the medium every 2 days after plating. If the cells do not reach confluency at the same time between different wells, discard the plate and repeat step 1.5.</li>
	<li>Once seeded, wait for the cells to reach 100% confluency within 3-5 days. Change the medium 24 h prior to the functional studies.</li>
</ol>
2. CFTR functional assay buffer solution preparation
<ol>
	<li>Prepare the sodium-free, chloride-free buffer solution with the reagents listed in Table 1.</li>
	<li>Add the reagents to tissue culture grade, double-distilled H2O (ddH2O). Allow the reagents to dissolve (in a closed container to avoid evaporation) by stirring overnight at room temperature.</li>
	<li>Once the solution is stable, measure the pH. If the pH of the solution exceeds 7.4, add 1 M N-methyl-D-glucamine (NMDG) solution dropwise to adjust the pH to 7.4. 
	NOTE: Do not adjust the pH of the solution with HCl because the solution must remain chloride-free.</li>
	<li>Allow the solution to mix for another 30 min and measure the osmolarity of the solution.
	<ol>
		<li>Reduce the osmolarity of buffer solution to a physiological range of 300 &#177; 5 mOsm/kg.</li>
	</ol>
	</li>
	<li>Filter and store the buffer solution in sterile bottles. 
	&#8203;NOTE: The solution can be stored at 4 &#176;C for up to 6 months. If stored for longer than 6 months, check the pH and osmolarity at room temperature before use.</li>
</ol>
3. CFTR functional assay
<ol>
	<li>Dissolve the membrane potential dye in the sodium-free, chloride-free buffer solution (0.5 mg of dye/1 mL of buffer solution) and warm it to 37 &#176;C. 
	NOTE: Avoid exposing the membrane potential dye powder to light.</li>
	<li>Remove the culture medium from Calu-3 or Caco-2 cell monolayers and add the dye-containing solution to each well (195 &#181;L per well for a 96-well plate, 95 &#181;L per well for a 384-well plate). 
	NOTE: Up to 112 &#181;L can be added per well of 384-well plates.</li>
	<li>Allow the cells to load the dye for 35 min at 37 &#176;C and 5% CO2.</li>
	<li>Move the cells, loaded with dye solution, to the fluorescence plate reader. Take fluorescence measurements from the bottom of the plate, with excitation = 530 &#177; 5 nm, emission = 560 &#177; 5 nm</li>
	<li>Begin by taking continuous baseline readings for 5 min at 30 s intervals.</li>
	<li>Add 5 &#181;L of a low-concentration solution of forskolin or DMSO to each well for a final concentration of 1 &#181;M forskolin (1x). Take a stimulation reading for 20 min with measurements at 15 s intervals.
	<ol>
		<li>For the 96-well plate format, prepare the low-concentration (40 &#181;M forskolin (1:25 dilution)) solution of forskolin by diluting 2 &#181;L of 1 mM forskolin (1,000x) stock solution or DMSO in 48 &#181;L of sodium-free, chloride-free buffer solution. 
		NOTE: The low-concentration 40 &#181;M forskolin is followed by a second dilution (1:40 dilution) in the next step for a final concentration of 1 &#181;M forskolin.</li>
		<li>For the 384-well plate format, prepare the low-concentration (20 &#181;M forskolin (1:50 dilution)) solution by diluting 1 &#181;L of 1 mM (1,000x) forskolin stock solution or DMSO in 49 &#181;L of sodium-free, chloride-free buffer solution. 
		NOTE: The low-concentration 20 &#181;M forskolin is followed by a second dilution (1:20 dilution) in the next step for a final concentration of 1 &#181;M forskolin.</li>
	</ol>
	</li>
	<li>Add 5 &#181;L of low-concentration solution of CFTRInh172 for a final concentration of 10 &#181;M CFTRInh172. Take an inhibition reading for 15 min with measurements at 30 s intervals.
	<ol>
		<li>For the 96-well plate format, prepare the CFTRInh172 low-concentration (400 &#181;M CFTRInh172 (1:25 dilution)) solution by diluting 2 &#181;L of a 10 mM CFTRInh172 (1,000x) stock solution in 48 &#181;L of sodium-free, chloride-free buffer solution. 
		NOTE: The low-concentration 400 &#181;M CFTRInh172 is followed by a second dilution (1:40 dilution) in the next step for a final concentration of10 &#181;M CFTRInh172.</li>
		<li>For the 384-well plate format, prepare the CFTRInh172 low-concentration (200 &#181;M CFTRInh172 (1:50 dilution)) solution by diluting 1 &#181;L of 10 mM CFTRInh172 (1,000x) stock solution in 49 &#181;L of sodium-free, chloride-free buffer solution. 
		&#8203;NOTE: The low-concentration 200 &#181;M CFTRInh172 is followed by a second dilution (1:20 dilution) in the next step for a final concentration of10 &#181;M CFTRInh172.</li>
	</ol>
	</li>
	<li>Quantify the fluorescence intensity measurements of each well, and export the values as a spreadsheet in column format containing individual wells. To calculate forskolin-induced changes, divide the RFU (Relative Fluorescence Units) measurements from each well of the 96- or 384-well plate by the last fluorescence intensity measurement of the baseline reading and plot them.
	<ol>
		<li>Alternatively, quantify CFTRInh172-mediated inhibition response to better confirm the specificity of CFTR function12, as acute treatment with forskolin may result in membrane depolarization through the activity of other chloride channels, such as SLC26A912.</li>
	</ol>
	</li>
	<li>Measure the peak responses as the maximum fluorescence intensity measurement from baseline during forskolin stimulation. Use this measurement or area under the curve to quantify the CFTR response.</li>
</ol>
4. ENaC functional assay buffer solution preparation
<ol>
	<li>Prepare the high-sodium, chloride-free buffer solution with the reagents listed in Table 2.</li>
	<li>Once the solution is stable, measure the pH. If the pH of the solution is below 7.4, add 1 N NaOH solution dropwise to adjust the pH to 7.4. 
	NOTE: Do not adjust the pH of the solution with HCl because the solution must remain chloride-free.</li>
	<li>Reduce the osmolarity of buffer solution to a physiological range of 300 &#177; 5 mOsm/kg.</li>
	<li>Filter and store the buffer solution in sterile bottles at 4 &#176;C for up to 6 months. 
	&#8203;NOTE: If storage is longer than 6 months, check the pH and osmolarity at room temperature before use.</li>
</ol>
5. ENaC functional assay
<ol>
	<li>Dissolve the membrane potential dye in the high-sodium, chloride-free buffer solution (0.5 mg of dye/1 mL of buffer solution) and warm it to 37 &#176;C. 
	NOTE: Avoid exposing the membrane potential dye powder to light.</li>
	<li>Remove the culture medium from Calu-3 or Caco-2 cell monolayers and add the dye-containing solution to each well (195 &#181;L per well for 96-well plates, 95 &#181;L per well for 384-well plates). 
	NOTE: Avoid exposing the dye solution to light.</li>
	<li>Allow the cells to load dye for 35 min at 37 &#176;C and 5% CO2.</li>
	<li>Move the cells, loaded with dye solution, to the fluorescence plate reader. Read fluorescence measurements from the bottom of the plate, with excitation = 530 &#177; 5 nm, emission = 560 &#177; 5 nm.</li>
	<li>Begin by taking continuous baseline readings for 5 min at 30 s intervals.</li>
	<li>To inhibit ENaC function, add 5 &#181;L of low-concentration solution amiloride or low-concentration solution DMSO dissolved in the high sodium, zero chloride buffer solution for a final concentration of 10 &#181;M amiloride.
	<ol>
		<li>For the 96-well plate format, prepare the low-concentration (400 &#181;M amiloride (1:25 dilution)) solution of amiloride by diluting 2 &#181;L of 10 mM amiloride (1,000x) stock solution in 48 &#181;L of high-sodium, chloride-free buffer solution. 
		NOTE: The low-concentration 400 &#181;M amiloride is followed by a second dilution (1:40 dilution) in the next step for a final concentration of 10 &#181;M amiloride.</li>
		<li>For the 384-well plate format, prepare the low-concentration (200 &#181;M amiloride (1:50 dilution)) solution of amiloride by adding 1 &#181;L of 10 mM amiloride (1,000x) stock solution in 49 &#181;L of high-sodium, chloride-free buffer solution. 
		NOTE: The low-concentration 200 &#181;M amiloride is followed by a second dilution (1:20 dilution) in the next step for a final concentration of 10 &#181;M amiloride.</li>
	</ol>
	</li>
	<li>Take an inhibition reading for 60 min with measurements at 45 s intervals.</li>
	<li>Repeat step 3.7 for data analysis.</li>
	<li>Measure the amiloride response as the percent inhibition from baseline to the point of plateau, approximately 15-20 min after amiloride addition. Use this difference to quantify the ENaC response.</li>
</ol>

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X., 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+platform+for+high-throughput+therapy+testing+on+iPSC-derived+lung+progenitor+cells+from+cystic+fibrosis+patients.">A new platform for high-throughput therapy testing on iPSC-derived lung progenitor cells from cystic fibrosis patients.</a> Stem Cell Reports. 16 (11), 2825-2837 (2021).</li><li>Hu, 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=Human+Embryonic+Kidney+293+Cells:+a+vehicle+for+biopharmaceutical+manufacturing,+structural+biology,+and+electrophysiology.">Human Embryonic Kidney 293 Cells: a vehicle for biopharmaceutical manufacturing, structural biology, and electrophysiology.</a> Cells, Tissues, Organs. 205 (1), 1-8 (2018).</li><li>Hadida, S., 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=Discovery+of+N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide+(VX-770,+ivacaftor),+a+potent+and+orally+bioavailable+CFTR+potentiator.">Discovery of N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (VX-770, ivacaftor), a potent and orally bioavailable CFTR potentiator.</a> Journal of Medicinal Chemistry. 57 (23), 9776-9795 (2014).</li><li>Chen, M. X., 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=Validation+and+optimization+of+novel+high-throughput+assays+for+human+epithelial+sodium+channels.">Validation and optimization of novel high-throughput assays for human epithelial sodium channels.</a> Journal of Biomolecular Screening. 20 (2), 242-253 (2015).</li><li>Maitra, R., Sivashanmugam, P., Warner, 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+rapid+membrane+potential+assay+to+monitor+CFTR+function+and+inhibition.">A rapid membrane potential assay to monitor CFTR function and inhibition.</a> Journal of Biomolecular Screening. 18 (9), 1132-1137 (2013).</li><li>Mall, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ENaC+inhibition+in+cystic+fibrosis:+potential+role+in+the+new+era+of+CFTR+modulator+therapies.">ENaC inhibition in cystic fibrosis: potential role in the new era of CFTR modulator therapies.</a> European Respiratory Journal. 56 (6), 2000946 (2020).</li><li>Moore, P. J., Tarran, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epithelial+sodium+channel+(ENaC)+as+a+therapeutic+target+for+cystic+fibrosis+lung+disease.">The epithelial sodium channel (ENaC) as a therapeutic target for cystic fibrosis lung disease.</a> Expert Opinion on Therapeutic Targets. 22 (8), 687-701 (2018).</li><li>Stutts, M. 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=CFTR+as+a+cAMP-dependent+regulator+of+sodium+channels.">CFTR as a cAMP-dependent regulator of sodium channels.</a> Science. 269 (5225), 847-850 (1995).</li><li>Merkert, S., 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=High-throughput+screening+for+modulators+of+CFTR+activity+based+on+genetically+engineered+cystic+fibrosis+disease-specific+iPSCs.">High-throughput screening for modulators of CFTR activity based on genetically engineered cystic fibrosis disease-specific iPSCs.</a> Stem Cell Reports. 12 (6), 1389-1403 (2019).</li><li>Bertrand, C. 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Lung Cellular and Molecular Physiology. 312 (6), 912-925 (2017).</li><li>Frelin, 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=Amiloride+and+its+analogs+as+tools+to+inhibit+Na++transport+via+the+Na++channel+the+Na+/H++antiport+and+the+Na+/Ca2++exchanger.">Amiloride and its analogs as tools to inhibit Na+ transport via the Na+ channel the Na+/H+ antiport and the Na+/Ca2+ exchanger.</a> Biochimie. 70 (9), 1285-1290 (1988).</li><li>Di Paola, M., 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=SLC6A14+is+a+genetic+modifier+of+cystic+fibrosis+that+regulates+Pseudomonas+aeruginosa+attachment+to+human+bronchial+epithelial+cells.">SLC6A14 is a genetic modifier of cystic fibrosis that regulates Pseudomonas aeruginosa attachment to human bronchial epithelial cells.</a> mBio. 8 (6), 02073 (2017).</li><li>Schiavi, S. 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=Biosynthetic+and+growth+abnormalities+are+associated+with+high-level+expression+of+CFTR+in+heterologous+cells.">Biosynthetic and growth abnormalities are associated with high-level expression of CFTR in heterologous cells.</a> American Journal of Physiology. 270, 341-351 (1996).</li><li>Ogawa, M., 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=Generation+of+functional+ciliated+cholangiocytes+from+human+pluripotent+stem+cells.">Generation of functional ciliated cholangiocytes from human pluripotent stem cells.</a> Nature Communications. 12 (1), 6504 (2021).</li><li>Lu, C., Pribanic, S., Debonneville, A., Jiang, C., Rotin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PY+motif+of+ENaC,+mutated+in+Liddle+syndrome,+regulates+channel+internalization,+sorting+and+mobilization+from+subapical+pool.">The PY motif of ENaC, mutated in Liddle syndrome, regulates channel internalization, sorting and mobilization from subapical pool.</a> Traffic. 8 (9), 1246-1264 (2007).</li></ol>

The authors declare no competing interests.

Here, we have described fluorescence-based methods for measuring CFTR and ENaC activity in the epithelial colorectal cancer cell line, Caco-2, and the human epithelial lung cancer cell line, Calu-3. These membrane potential assays in epithelial cell lines can be used to confirm the efficacy of small molecule modulator compounds, previously identified in heterologous expression systems, prior to final in vitro validation in primary patient-derived epithelial cultures. 
It is imperative to confirm that the above measurements are appropriately attributed to the channel of interest. For example, the measurement ascribed to CFTR should show dependence on CFTR protein expression and the electrochemical driving force of chloride, which is modulated by forskolin and the inhibitor CFTRInh-172. Similarly, the membrane potential changes caused by 10 &#181;M amiloride can be attributed to ENaC, if they are reliant on ENaC protein expression and an inward sodium driving force. Although we have confirmed these properties of ENaC in MDCK cells following transfection with all three subunits of ENaC2, we have not yet formally confirmed that the amiloride response measured in Caco-2 and Calu-3 cells is conferred by ENaC. This is particularly important, as amiloride can modulate the function of the sodium-proton exchanger NHE3 in addition to ENaC13. Conceivably, the amiloride-induced hyperpolarization observed in this assay could partially reflect the indirect effects of intracellular acidification on other channels. To confirm that the amiloride-induced hyperpolarization measured using this membrane potential-sensitive dye is conferred by ENaC in the above cell lines and more complex cell systems, such as iPSC-derived tissues, we are confirming that this activity is lost by knocking out an obligate ENaC subunit (beta) in these lines. 
The success of these assays requires attention to several factors. First, the epithelial cultures must be confluent and well differentiated. For the measurement of CFTR and ENaC function, approximately 145,000 cells should be plated per well in 96-well plates or 45,000 cells per well in 384-well plates. Once the epithelial cells reach confluency (within 3-4 days from plating), allow 2-5 days of differentiation. This timing was determined previously based on maximal CFTR protein expression by immunoblotting14. Similarly, optimal timing was determined for functional ENaC expression in both cell lines, Calu-3 and Caco-2. 
Nonconfluent monolayers or cell overgrowth leads to low reproducibility across technical replicates. In cases where cells in individual wells do not reach confluency at the same time, these plates should be discarded. Additionally, unstable baseline readings or lack of stimulation responses may be an indication of poor cell quality, which must be excluded from analysis. Reduced responses to the CFTR activator, forskolin, or the ENaC inhibitor, amiloride, could potentially reflect the use of excessively passaged cells15. Although not an issue for Calu-3 and Caco-2 cells, other cells or tissues may not adhere well to the plates and may require precoating of the wells. For example, in previous studies, 2D cholangiocyte cultures were attached to the plates using collagen coating16. Alternatively, the adherence of 2D intestinal tissues was increased using Poly-L-Lysine2. 
Further, in testing the modulation of ion channels by small molecules using a fluorescence-based assay, it is critical that potential artifacts conferred by fluorescent properties of the small molecules be addressed. To confirm the specificity of functional responses, membrane potential assays can be further validated using conventional electrophysiological methods, such as Ussing studies17. 
After confirming that the response detected by membrane potential-sensitive dyes is specifically conferred by the channel of interest, these assays have tremendous potential to validate promising modulators of epithelial ion channels in their relevant cellular context. This platform can bridge the gap between high-throughput modulator screens in heterologous expression systems and time-consuming, bioelectric measurements in difficult-to-access primary tissues.

Fluorescence-based, high-throughput activity assays of recombinant channel proteins have been highly effective in identifying small-molecule modulators that have the potential to become future therapies1,2,3,4. One common method of screening small molecule libraries for ion channel modulators is through measuring changes in membrane potential. Typically, these screens are conducted using recombinant channels expressed heterologously in cell lines such as HEK-293 cells5,6,7.
However, there is a need for validation of &#34;hits&#34; in relevant cell types. We propose that a secondary screen in relevant cell lines that endogenously express the channels of interest is desirable. Such a secondary screen will identify those modulators most likely to be effective in final validation assays that rely on less accessible primary human tissues.
There is also the need for assay systems that have the flexibility to report the activity of multiple ion channel targets in the same tissue type. For example, there is substantive published literature that supports the concept that CFTR and ENaC functionally interact8,9,10. Although several well-studied epithelial cell lines are known to express both CFTR and ENaC, to date, the assay conditions for studying both in a high-throughput manner have not been described.
This fluorescence-based membrane potential assay is versatile, as it employs an exogenous chemical probe to measure the function of CFTR and ENaC, bypassing the need for genetically encoded probes11. As proof of principle, two commonly used epithelial cell lines are studied as examples to highlight the critical steps necessary for measuring channel activity of both CFTR and ENaC.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amiloride</td>
			<td>Spectrum Chemical</td>
			<td>TCI-A2599-5G</td>
			<td>Dissolved in DMSO and stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>CFTRInh-172</td>
			<td>CF Foundation Therapeutics</td>
			<td></td>
			<td>Dissolved in DMSO and stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>EMEM, 1xs</td>
			<td>Wisent</td>
			<td>320-005-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Wisent</td>
			<td>080-450</td>
			<td></td>
		</tr>
		<tr>
			<td>FLIPR Membrane Potential Dye</td>
			<td>Molecular Devices</td>
			<td>R8042</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma-Aldrich</td>
			<td>F3917</td>
			<td>Dissolved in DMSO and stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Gluconic acid lactone or (D-(+)-Gluconic acid &#948;-lactone)</td>
			<td>Sigma-Aldrich</td>
			<td>G4750</td>
			<td>Stored at room temperature</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Bioshop</td>
			<td>HEP001.5</td>
			<td>Stored at room temperature</td>
		</tr>
		<tr>
			<td>Human Bronchial Adenocarcinoma Cell Line (Calu-3)</td>
			<td>ATCC</td>
			<td>HTB-55</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Epithelial Colorectal Adenocarcinoma Cell Line (Caco-2)</td>
			<td>ATCC</td>
			<td>HTB-37</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Methyl-D-glucamine (NMDG</td>
			<td>Sigma-Aldrich</td>
			<td>M2004</td>
			<td>Stored at room temperature</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution</td>
			<td>Wisent</td>
			<td>450-200-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Wisent</td>
			<td>311-010-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Gluconate</td>
			<td>Sigma-Aldrich</td>
			<td>P1847</td>
			<td>Stored at room temperature</td>
		</tr>
		<tr>
			<td>Sodium Gluconate</td>
			<td>Sigma-Aldrich</td>
			<td>G9005</td>
			<td>Stored at room temperature</td>
		</tr>
	</tbody>
</table>

a fluorescence-based assay of membrane potential for high-throughput functional study of two endogenous ion channels in two epithelial cell lines

Fluorescence-based studies are suitable for high-throughput plate reader assays of cells in culture. They have been commonly employed for drug discovery campaigns targeting recombinant ion channel proteins overexpressed in cells such as HEK-293 cells. However, there is increasing emphasis on the use of tissue-relevant cell lines for studying the effects of small molecule interventions. The following protocol describes the adaptation of a fluorescence-based membrane potential assay for the study of ion channels endogenously expressed in epithelial cell lines. The membrane potential assay details a high-throughput assay for chloride channel activity of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in two commonly studied epithelial cell lines, Caco-2 and Calu-3. In addition, this paper describes a novel application of this system to measure the activity of the Epithelial Sodium Channel (ENaC) in a high-throughput format in the same epithelial cell lines. Together, these fluorescence-based assays provide a robust and flexible platform for studying small molecule modulators, targeting two epithelial channels in a relevant cellular context.

To measure CFTR channel activity, well-differentiated, adhered cells are incubated in a zero-chloride extracellular solution containing the membrane potential-sensitive dye. CFTR function is consistently detected as membrane depolarization after forskolin stimulation. Chloride efflux is detected as a sharp increase in fluorescence compared to the vehicle control. The fluorescence signal is sustained until the addition of CFTR inhibitor (CFTRInh172), leading to a rapid decline in the fluorescence intensity, as seen in Caco-2 cells (Figure 1A), and is reproducible in both Caco-2 and Calu-3 cells (Figure 1B). The assessment of CFTR activity as the difference in the maximal change in fluorescence after forskolin or the DMSO (vehicle). Individual points ranged between &#177; 3 standard deviations from the mean forskolin stimulation (black points) or DMSO control (grey points), which corresponded to a Z&#39; factor of 0.586. This excellent score indicated the reproducibility of this assay (Figure 1C). In case there is a minor drift in the baseline, the impact of the drift can be minimized by extrapolating the baseline throughout the trace after activation up to the point of inhibitor addition. The response can be quantified as the area under the curve, with the lower limit defined by the extrapolated line.
ENaC activity can also be measured in confluent airway and colonic epithelial cells grown in 96-well plates. In contrast to the assay for CFTR channel activity, these cells are submerged in a high-sodium-containing solution, enabling sodium absorption through ENaC. With the acute addition of amiloride, sodium uptake is blocked, and cells experience relative membrane hyperpolarization. This is shown in Caco-2 cells at both low (10 &#181;M) (Figure 2A) and high concentrations (50 &#181;M) of amiloride (Figure 2A,B) and in Calu-3 cells with a high concentration of amiloride (50 &#181;M) (Figure 2B). The ENaC assay was expanded to a 384-well plate format and demonstrated great reproducibility in ENaC inhibition. Similarly, the individual points ranged between &#177; 3 standard deviations from the mean amiloride inhibition (black points) or DMSO control (grey points). The ENaC response in Calu-3 cells reported a Z&#39; factor of 0.629 with acute treatment using amiloride (50 &#181;M) (Figure 2C), which indicates that these parameters support high-throughput screening.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63528/63528fig01.jpg" /> 
Figure 1: CFTR channel measurement as changes in membrane depolarization in Caco-2 and Calu-3 cells. (A) Representative trace of CFTR stimulation with forskolin (1 &#181;M)and DMSO control in Caco-2 cells. (B) Box and whisker plot of forskolin stimulation in Caco-2 and Calu-3 cells (**** P &#60; 0.0001, n &#62; 3 biological replicates, n &#62; 3 technical replicates). Biological replicates are independent cell line passages; technical replicates refer to independent wells of the multiwell plates. Box plot depicts the median value; bounds depict IQR, with the whiskers defining the minima and maxima values. (C) Bland-Altman plot of reproducible CFTR response with forskolin stimulation compared to DMSO control in Caco-2 cells. Black points represent maximal absolute changes in fluorescence in response to forskolin stimulation. Grey points represent changes in fluorescence in response to DMSO control. Abbreviations: CFTR = Cystic Fibrosis Transmembrane Conductance Regulator; DMSO = dimethyl sulfoxide; IQR = interquartile range; Fsk = forskolin; &#916;F/F0 = change in fluorescence. <a href="https://www.jove.com/files/ftp_upload/63528/63528fig01large.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/63528/63528fig02.jpg" /> 
Figure 2: ENaC inhibition measurements through membrane hyperpolarization in Caco-2 and Calu-3 cells. (A) Representative trace of ENaC inhibition with amiloride (10 &#181;M)relative to DMSO control in Caco-2 cells. (B) Box and whisker plot of amiloride inhibition in Caco-2 and Calu-3 cells (**** P &#60; 0.0001, n &#62; 3 biological replicates, n &#62; 3 technical replicates). (C)&#160;Bland-Altman plot of reproducible ENaC response with amiloride inhibition compared to DMSO control in Calu-3 cells. Black points represent maximum absolute changes in fluorescence in response to amiloride inhibition. Grey points represent changes in fluorescence in response to DMSO control. Abbreviations: ENaC = epithelial sodium channel; DMSO = dimethyl sulfoxide; Amil. = amiloride; &#916;F/F0 = change in fluorescence. <a href="https://www.jove.com/files/ftp_upload/63528/63528fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>NMDG (N-Methyl-D-glucamine)</td>
			<td>150 mM</td>
		</tr>
		<tr>
			<td>Gluconic Acid Lactone</td>
			<td>150 mM</td>
		</tr>
		<tr>
			<td>Potassium Gluconate</td>
			<td>3 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>10 mM</td>
		</tr>
	</tbody>
</table>
Table 1: Reagents and their corresponding concentrations for CFTR functional assay with zero sodium and chloride. Abbreviation:&#160;CFTR = Cystic Fibrosis Transmembrane Conductance Regulator.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>Sodium Gluconate</td>
			<td>150 mM</td>
		</tr>
		<tr>
			<td>Potassium Gluconate</td>
			<td>3 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>10 mM</td>
		</tr>
	</tbody>
</table>
Table 2: Reagents and their corresponding concentrations for ENaC functional assay buffer with high sodium, zero chloride.&#160;Abbreviation: ENaC = Epithelial sodium (Na) Channel.

Watch this Scientific Journal Video about A Fluorescence-Based Assay of Membrane Potential for High-Throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines at JoVE.com

A Fluorescence-Based Assay of Membrane Potential for High-Throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines

This protocol describes a method for the study of electrogenic membrane proteins by measuring changes in membrane potential. This assay provides a platform for the functional readout of multiple ion channels endogenously expressed in epithelial cell lines.

Fluorescence-based studies are suitable for high-throughput plate reader assays of cells in culture. They have been commonly employed for drug discovery ...

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1.9K Views.  Hospital for Sick Children. This protocol describes a method for the study of electrogenic membrane proteins by measuring changes in membrane potential. This assay provides a platform for the functional readout of multiple ion channels endogenously expressed in epithelial cell lines. 

Video: A Fluorescence-Based Assay of Membrane Potential for High-Throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...