This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2011-0023701, 2016R1D1A1A02937397, and 2018R1A6A1A03023718).

1. Generation of lentiviruses expressing YFPQL and SLC26A4
<ol>
	<li>Grow HEK293T human embryonic kidney cells to 80% confluency on 100 mm culture plates. Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 &#181;g/mL streptomycin is the culture medium used throughout the protocol to maintain HEK293T and other cells mentioned below.</li>
	<li>Coat 6-well culture plates by adding 2 mL of 0.005% of sterile poly-L-lysine (PLL) solution to each well for 10 min. Aspirate the PLL solution and rinse the surface twice with 2 mL of sterile water.</li>
	<li>Wash the HEK293T cells with 10 mL of phosphate buffered saline (PBS) and treat the cell monolayers in each 100 mm dish with 2 mL of 0.25% trypsin-EDTA solution at 37 &#176;C for 3 min. Add 5 mL of culture medium and resuspend the cells.</li>
	<li>Count the cells in a hemocytometer and adjust the density of the cell suspension to 250,000 cells/mL in culture medium and add 500,000 cells in 2 mL of culture medium to each lysine-coated well of 6-well plates. Incubate the cells in a humidified 5% CO2/95% air atmosphere at 37 &#176;C for 24 h and then replace the culture medium with DMEM without penicillin or streptomycin.</li>
	<li>In a 1.5 mL tube, dilute 20 &#181;L of transfection reagent with 500 &#181;L of DMEM without serum or antibiotics. Mix gently by pipetting and let stand at room temperature for 5 min.</li>
	<li>Meanwhile, pipette 250 &#181;L of DMEM into each of two 1.5 mL tubes and then add 1500 ng of pLVX-EIP-YFPQL or pLenti6P-SLC26A4, 1225 ng of psPAX2 and 375 ng of pMD2.G to each. The two lentiviral plasmids have been previously described21. Add 250 &#181;L of diluted transfection reagent to each plasmid tube, mix gently and incubate for 20 min at room temperature.</li>
	<li>After 20 min, add 500 &#181;L of transfection reagent and plasmid complexes in the 1.5 mL tubes dropwise to each culture plate well in step 1.4 and mix by rocking the plate back and forth. Incubate cells at 37 &#176;C in a CO2 incubator for 12 h.</li>
	<li>Replace the medium with 2.5 mL of fresh medium and incubate for an additional 48 h. Then place the culture plate on ice for 5 min to keep the conditioned medium containing lentivirus chilled to maintain infectivity.</li>
	<li>Harvest the media containing lentiviruses and transfer to 15 mL conical tubes. Centrifuge at 3,000 x g at 4 &#176;C for 3 min and then remove floating HEK293T cells from the supernatant by filtration at 0.4 &#181;m.</li>
	<li>Store the media containing lentiviruses at 4 &#176;C for use within 2 days. For later use, store 200 &#181;L aliquots at &#8722;80 &#176;C.</li>
</ol>
2. Generation of LN215-YFPQL and LN215-I&#8722; cells by lentiviral transduction
<ol>
	<li>Grow LN215 cells in 100 mm culture plates to 80% confluency in DMEM supplemented with 10% fetal bovine serum (FBS) 100 U/mL penicillin, and 100 &#181;g/mL streptomycin as described above. 
	NOTE: If the I-YFP-GJIC assay is conducted using a different cell line, use the appropriate culture medium. LN215-YFPQL, and LN215-I- cells can be provided by the University-Industry foundation, Yonsei University. Please contact the corresponding author.</li>
	<li>One day before transduction, wash the cells twice with 10 mL of PBS, treat with 2 mL of 0.25% trypsin-EDTA at 37 &#176;C for 3 min. Resuspend the cells in 5 mL of culture medium with a 10 mL serologic pipette and adjust the density to 50,000 cells/mL. Add 20,000 cells in 400 &#181;L of media to each well of a 24-well culture plate for treatment as no virus control, YFPQL, and SLC26A4 cells.</li>
	<li>After 24 h of incubation at 37 &#176;C, transduce two wells by replacing the culture medium with 400 &#181;L of a 1:1 mixture of pLVX-EIP-YFPQL or pLenti6P-SLC26A4 lentivirus and fresh culture medium supplemented with polybrene at a final concentration of 4 &#181;g/mL. For no virus controls, replace with culture medium.</li>
	<li>Incubate the cells at 37 &#176;C for 15 h, aspirate the medium containing lentiviruses, add fresh culture medium, and incubate the cells for an additional 72 h. 
	CAUTION: To prevent contamination of lentivirus between wells, use new tips or pipets for each well when you aspirate culture medium containing lentivirus or dispense fresh growth medium.</li>
	<li>Wash the cells in each well twice with 0.5 mL of PBS, treat with 300 &#181;L of trypsin-EDTA for 3 min. Resuspend the cells in 2 mL of culture medium and plate in six-well culture plates with 2 &#181;g/mL puromycin.</li>
	<li>Culture the cells in media containing 2 &#181;g/mL puromycin until all cells in the control well are dead (round-shaped or floating when observed in microscope), which usually takes a week. Refresh the culture media containing puromycin every other day during the selection period. If LN215-YFPQL or LN215-I- cultures become confluent before selection has completed, transfer the cells to 100 mm plate and continue selection as in step 2.5.</li>
</ol>
3. Preparation of solutions required for the assay
<ol>
	<li>Prepare 500 mL of C-solution (10 mM HEPES, 140 mM NaCl, 10 mM glucose, 5 mM KCl, 1 mM MgCl2, and 1 mM CaCl2) and 500 mL of I-solution (10 mM HEPES, 140 mM NaI, 10 mM glucose, 5 mM KCl, and 1 mM CaCl2).</li>
	<li>Adjust the pH of both solutions to 7.4 with 1 N NaOH, sterilize the solutions by filtrations at 0.4 &#181;m for storage. Store at 4 &#176;C for up to 1 month. Check the pH before using.</li>
</ol>
4. Plating the LN215-YFPQL and LN215-I&#8722; cells
<ol>
	<li>Culture LN215-YFPQL and LN215-I&#8722; cells in 100 mm plates separately in culture medium to reach the populations required for assay. LN215-YFPQL and LN215-I&#8722; cells in 40% and 80% confluency in 100 mm plates, respectively, are sufficient for a 96-well plate assay.</li>
	<li>One day before conducting the I-YFP GJIC assay, wash each 100 mm culture plate with 10 mL of PBS. Treat each plate with 2 mL of 0.25% trypsin-EDTA solution and incubate at 37 &#176;C for 5 min. Resuspend the cells in each plate in 4 mL of culture medium and transfer to 15 mL conical tubes.</li>
	<li>Pellet the cells by centrifugation at 1,000 x g for 3 min. Discard the supernatant and resuspend each cell pellet with 5 mL of culture medium. Break up any cell clumps into single cells by pipetting up and down about 20 times with a 10 mL serological pipette.</li>
	<li>Count the cells in a hemocytometer, and dilute the cells in the culture medium to make cell suspensions of LN215-YFPQL at 80,000 cells/mL and LN215-I&#8722; at 160,000 cells/mL.</li>
	<li>Mix 7 mL of LN215-YFPQL and 7 mL of LN215-I&#8722; cell suspensions in a reservoir. Add 100 &#181;L of the mixture to each well of a 96-well cell culture plate using a multichannel pipette. 
	NOTE: To add 100 &#181;L of the mixture in each well of 96-well cell plate, about 10 mL of mixed cell suspension is needed. It is recommended to make more cell suspension than needed.</li>
	<li>Incubate the cells in humidified 5% CO2/95% air at 37 &#176;C for 24 h. The LN215-YFPQL and LN215-I&#8722;cell culture should be 100% confluent when the assay is conducted.</li>
</ol>
5. Conducting the I-YFP&#160;GJIC&#160;assay
NOTE: Use a fluorescence microscope with 20x magnification, and a GFP filter to check the 96-well plates to be sure there are no clumps of LN215-YFPQL or LN215-SLC26A4 cells and that the cell cultures are fully confluent and well distributed before conducting the assay.
<ol>
	<li>At least 30 min before doing the assay, turn on a microplate and set to 37 &#176;C.</li>
	<li>Wash the tubing of an automated injector with 3 mL of 70% ethanol, 3 mL of distilled water, and then 3 mL of I-solution at a flow rate of 300&#160;&#181;L/s.</li>
	<li>Warm the C- and I-solutions to 37 &#176;C in the water bath. 
	NOTE: As 100 &#181;L of each solution is needed for each well of the 96-well plate, about 10 mL of each solution is needed for each assay. An additional 10 mL of the I-solution is needed for priming each plate (total of 20 mL) and an additional 25 mL of the C-solution is needed for washing each plate (total of 35 mL).</li>
	<li>Aspirate the growth medium or invert the plate to empty it; tap out residual medium. 
	NOTE: Residual fetal bovine serum in the growth media causes background fluorescence and a decline in assay quality.</li>
	<li>Add 200 &#181;L of C-solution to each well from a reservoir using a multichannel pipette. Aspirate the C-solution or invert the plate to empty and tap out residual solution.</li>
	<li>Add 50 &#181;L &#181;L C-solution, 1 &#181;L of 2.5 mM chemical stock (see Table 1) or dimethylsulfoxide (DMSO) as a vehicle, and then 50 &#181;L &#181;L C-solution to each well with a multichannel pipette. 
	NOTE: Most reagents in a chemical library are dissolved in DMSO and up to 1% (v/v) is allowed in most cell-based assays24. As DMSO has a higher density than water, reagents dissolved in DMSO tend to go down to the bottom when added to the culture plate wells, which disturbs the concentrations of the assay solutions. This can be circumvented by adding 50 &#181;L of C-solution, reagents in DMSO, and 50 &#181;L C of -solution in order. The last 50 &#181;L of C-solution is for mixing.</li>
	<li>Incubate the cells at 37 &#176;C in air, not in 5% CO2. The incubation time can be modified, but 10 min is usually sufficient for ion-channel modulators to act.</li>
	<li>During incubation, set the microplate reader program to inject 100 &#181;L of I-solution to each well at 1 s and to measure the fluorescence for 10 s at 0.4 s intervals. Set the reader to read fluorescence from the bottom. The recommended injection speed is 135 &#181;L/s. Set the excitation wavelength to 485 nm and read the emission at 520 nm. 
	NOTE: Detailed settings for the microplate reader programs are as follows.
	<ol>
		<li>Click the Manage protocols button.</li>
		<li>Click the New Button. Select the Fluorescence Intensity in the measurement method section, and Well Mode in the reading mode section. Next, click the OK button. New tab will appear.</li>
		<li>In the Basic Parameters menu, set the excitation wavelength to 485 nm and the emission at 520 nm. Select the Bottom Optic to read fluorescence from the bottom. Set measurement start time to be &#34;0 s&#34;, number of intervals to be &#34;25&#34;, number of flashes per well, and interval to be &#34;20&#34;, and interval time to be &#34;0.4 s&#34;.</li>
		<li>In the Layout menu, draw region of the plate to be read.</li>
		<li>In the Concentrations/Volumes/Shacking menu, set the microplate reader to inject 100 &#181;L of I-solution to each well with 135 &#181;L/s injection speed. 
		NOTE: Avoid faster injection speeds because they can result in detachment of cells.</li>
		<li>In the Injection time menu, set the injection start time to be 1 s. Then, click the Start Measurement button.</li>
	</ol>
	</li>
	<li>After incubation, place the 96-well plates in the microplate reader and start the measurement by clicking Start Measurement button again.</li>
</ol>
6. Calculation of GJIC activity
<ol>
	<li>Calculate the percentages of YFPQL quenching and GJIC activity as23 
	YFPQL quenching (%) = <img alt="Equation 1" src="/files/ftp_upload/58966/58966eq1.jpg" /> 
	GJIC activity (%) = <img alt="Equation 2" src="/files/ftp_upload/58966/58966eq2.jpg" /> 
	NOTE: In principle, GJIC activity should be calculated from the difference of the percentages of YFP fluorescence in wells with acceptor cells and donor cells and the corresponding acceptor-cell only wells. However, as LN215-YFPQL cells show negligible YFP quenching by iodide after 10 s, we do not take the background YFP quenching into account when conducting HTS using LN215-YFPQL and LN215-I- cells.</li>
</ol>

<ol>
	<li>Goodenough, D. a., Goliger, J. a., Paul, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexins,+connexons,+and+Intercellular+Communication.">Connexins, connexons, and Intercellular Communication.</a> Annual Review of Biochemistry. 65, 475-502 (1996).</li><li>Upham, B. L., Weis, L. M., Trosko, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulated+Gap+Junctional+Intercellular+Communication+as+a+Biomarker+of+PAH+Epigenetic+Toxicity:+Structure-Function+Relationship.">Modulated Gap Junctional Intercellular Communication as a Biomarker of PAH Epigenetic Toxicity: Structure-Function Relationship.</a> Environmental Health Perspectives. 106, 975 (1998).</li><li>U, J. E. T., Chang, C., Upham, B., Wilson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+toxicology+as+toxicant-induced+changes+in+intracellular+signalling+leading+to+altered+gap+junctional+intercellular+communication.">Epigenetic toxicology as toxicant-induced changes in intracellular signalling leading to altered gap junctional intercellular communication.</a> Toxicology Letters. , 71-78 (1998).</li><li>Yamasaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+disrupted+gap+junctional+intercellular+communications+in+detection+and+characterization+of+carcinogens.">Role of disrupted gap junctional intercellular communications in detection and characterization of carcinogens.</a> Mutation Research - Reviews in Genetic Toxicology. , (1996).</li><li>Yamasaki, H., 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=Gap+junctional+intercellular+communication+and+cell+proliferation+during+rat+liver+carcinogenesis.">Gap junctional intercellular communication and cell proliferation during rat liver carcinogenesis.</a> Environmental Health Perspectives. 101, 191-197 (1993).</li><li>Vinken, 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=Gap+junctional+intercellular+communication+as+a+target+for+liver+toxicity+and+carcinogenicity.">Gap junctional intercellular communication as a target for liver toxicity and carcinogenicity.</a> Critical Reviews in Biochemistry and Molecular Biology. 44, 201-222 (2009).</li><li>Fonseca, C. G., Green, C. R., Nicholson, L. F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upregulation+in+astrocytic+connexin+43+gap+junction+levels+may+exacerbate+generalized+seizures+in+mesial+temporal+lobe+epilepsy.">Upregulation in astrocytic connexin 43 gap junction levels may exacerbate generalized seizures in mesial temporal lobe epilepsy.</a> Brain Research. 929 (1), 105-116 (2002).</li><li>Garbelli, 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=Expression+of+connexin+43+in+the+human+epileptic+and+drug-resistant+cerebral+cortex.">Expression of connexin 43 in the human epileptic and drug-resistant cerebral cortex.</a> Neurology. 76 (10), 895-902 (2011).</li><li>Schulz, 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=Connexin+43+is+an+emerging+therapeutic+target+in+ischemia/reperfusion+injury,+cardioprotection+and+neuroprotection.">Connexin 43 is an emerging therapeutic target in ischemia/reperfusion injury, cardioprotection and neuroprotection.</a> Pharmacology and Therapeutics. 153, 90-106 (2015).</li><li>Sarrouilhe, D., Dejean, C., Mesnil, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+gap+junction+channels+in+the+pathophysiology+of+migraine+with+aura.">Involvement of gap junction channels in the pathophysiology of migraine with aura.</a> Frontiers in Physiology. , (2014).</li><li>Patel, S. 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=Gap+junction+inhibition+prevents+drug-induced+liver+toxicity+and+fulminant+hepatic+failure.">Gap junction inhibition prevents drug-induced liver toxicity and fulminant hepatic failure.</a> Nature Biotechnology. 30 (2), 179-183 (2012).</li><li>Kandyba, E. E., Hodgins, M. B., Martin, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+living+skin+equivalent+amenable+to+live-cell+imaging:+analysis+of+the+roles+of+connexins+in+the+epidermis.">A murine living skin equivalent amenable to live-cell imaging: analysis of the roles of connexins in the epidermis.</a> Journal of Investigative Dermatology. 128 (4), 1039-1049 (2008).</li><li>Upham, B. 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=Differential+roles+of+2+,+6+,+and+8+carbon+ceramides+on+the+modulation+of+gap+junctional+communication+and+apoptosis+during+carcinogenesis.">Differential roles of 2 , 6 , and 8 carbon ceramides on the modulation of gap junctional communication and apoptosis during carcinogenesis.</a> Cancer Letters. 191, 27-34 (2003).</li><li>Upham, B. 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=Structure-activity-dependent+regulation+of+cell+communication+by+perfluorinated+fatty+acids+using+in+vivo+and+in+vitro+model+systems.">Structure-activity-dependent regulation of cell communication by perfluorinated fatty acids using in vivo and in vitro model systems.</a> Environmental Health Perspectives. 117 (4), 545-551 (2009).</li><li>Weis, L. 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=Bay+or+Baylike+Regions+of+Polycyclic+Aromatic+Hydrocarbons+Were+Potent+Inhibitors+of+Gap+Intercellular+Communication.">Bay or Baylike Regions of Polycyclic Aromatic Hydrocarbons Were Potent Inhibitors of Gap Intercellular Communication.</a> Environmental Health Perspectives. 106 (1), 17-22 (1998).</li><li>el-Fouly, M. H., Trosko, J. E., Chang, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scrape-Loading+and+Dye+Transfer:+A+Rapid+and+Simple+Technique+to+Study+Gap+Junctional+Intercellular+Communication.">Scrape-Loading and Dye Transfer: A Rapid and Simple Technique to Study Gap Junctional Intercellular Communication.</a> Experimental Cell Research. 168 (2), 422-430 (1987).</li><li>Wade, M. H., Trosko, J. E., Schindler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobleaching+Assay+of+Gap+Junction-+Mediated+Communication+Between+Human+Cells.">Photobleaching Assay of Gap Junction- Mediated Communication Between Human Cells.</a> Advancement of Science. 232 (4749), 525-528 (2010).</li><li>Abbaci, 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=In+vitro+characterization+of+gap+junctional+intercellular+communication+by+gap-FRAP+technique.">In vitro characterization of gap junctional intercellular communication by gap-FRAP technique.</a> Proceedings of SPIE. , 585909 (2005).</li><li>Neyton, J., Trautmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-channel+currents+of+an+intercellular+junction.">Single-channel currents of an intercellular junction.</a> Nature. 317 (6035), 331-335 (1985).</li><li>Wilders, R., Jongsma, 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=Limitations+of+the+dual+voltage+clamp+method+in+assaying+conductance+and+kinetics+of+gap+junction+channels.">Limitations of the dual voltage clamp method in assaying conductance and kinetics of gap junction channels.</a> Biophysical Journal. 63 (4), 942-953 (1992).</li><li>Lee, J. Y., Choi, E. J., Lee, J. <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+high-throughput+screening-compatible+gap+junctional+intercellular+communication+assay.">A new high-throughput screening-compatible gap junctional intercellular communication assay.</a> BMC Biotechnology. 15 (1), 1-9 (2015).</li><li>Galietta, L. J. V., Haggie, P. M., Verkman, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein-based+halide+indicators+with+improved+chloride+and+iodide+affinities.">Green fluorescent protein-based halide indicators with improved chloride and iodide affinities.</a> FEBS Letters. 499 (3), 220-224 (2001).</li><li>Lee, J. Y., Yoon, S. M., Choi, E. J., Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terbinafine+inhibits+gap+junctional+intercellular+communication.">Terbinafine inhibits gap junctional intercellular communication.</a> Toxicology and Applied Pharmacology. 307, 102-107 (2016).</li><li>Hughes, J., Rees, S., Kalindjian, S., Philpott, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+early+drug+discovery.">Principles of early drug discovery.</a> British Journal of Pharmacology. 162 (6), 1239-1249 (2011).</li><li>Zhang, J. H., Chung, T. D. Y., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+statistical+parameter+for+use+in+evaluation+and+validation+of+high+throughput+screening+assays.">A simple statistical parameter for use in evaluation and validation of high throughput screening assays.</a> Journal of Biomolecular Screening. 4 (2), 67-73 (1999).</li><li>Upham, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Integrative+Signaling+Through+Gap+Junctions+in+Toxicology.">Role of Integrative Signaling Through Gap Junctions in Toxicology.</a> Current Protocols in Toxicology. , (2011).</li><li>Schalper, K. 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=Modulation+of+gap+junction+channels+and+hemichannels+by+growth+factors.">Modulation of gap junction channels and hemichannels by growth factors.</a> Molecular BioSystems. 8 (3), 685-698 (2012).</li><li>Kulkarni, G. V., Mcculloch, C. A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+deprivation+induces+apoptotic+cell+death+in+a+subset+of+Balb+/+c+3T3+fibroblasts.">Serum deprivation induces apoptotic cell death in a subset of Balb / c 3T3 fibroblasts.</a> Journal of Cell Science. 1179, 1169-1179 (1994).</li><li>Harris, A. L., Locke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+of+Connexin+Channels.">Permeability of Connexin Channels.</a> Connexins. , 165-206 (2009).</li><li>Choi, E. J., Yeo, J. H., Yoon, S. M., Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gambogic+Acid+and+Its+Analogs+Inhibit+Gap.">Gambogic Acid and Its Analogs Inhibit Gap.</a> Junctional Intercellular Communication. 9, 1-10 (2018).</li></ol>

The authors have no conflicts of interest to disclose.

The I-YFP-GJIC assay can be used for HTS because it is robust, rapid, and inexpensive. An HTS assay is considered robust if the Z&#39;-factor is above 0.525. See Zhang et al. for a description of the statistical analysis used to assessing the suitability of HTS assays25. When LN215 cells were used, the Z&#39;-factor was &#62;0.5 without any assay optimization. If other cell types are used in the assay and its Z&#39;-factor is &#60;0.5, the robustness can be improved by extending the assay time21. LN215 and HOS, human osteosarcoma cells, cells need only 10 s to obtain a Z&#39;-factor &#62;0.521. The assay requires only acceptor and donor cells, the I-solution, and the C-solution. No additional reagents, such as LY and calcein-AM are needed. Another advantage of this GJIC assay is that it measures the total GJIC activity of the cells in a single well, which results in low between-well variability. In microinjection, gap-fluorescence recovery after photobleaching (FRAP), and dual patch clamp assays, a cell in the assayed area may significantly affect the measurement. Although scrape loading assays evaluate GJIC over a relatively wide area, the scraping-loading procedure can introduce significant variability26.
The critical steps for successful conduct of the I-YFP-GJIC assay are as follows. The pH of the C- and I-solutions must be adjusted to 7.4 before each use. YFPQL fluorescence is strongly affected by pH as well as by halides22. A pH difference between the two solutions can lead to aberrant change in YFP fluorescence after addition of the I-solution. The donor and acceptor cells must be completely dissociated before plating. Clumps of donor or acceptor cells disturb the assay. The confluence of the mixed culture should be 100% to maximize formation of GJs between cells. The ratio of acceptor to donor cells also affects the assay results as described by Lee et al.21. As only acceptor cells have YFP fluorescence, the ratio and arrangement of acceptor and donor cells can be easily checked with a fluorescence microscope before conducting the assay. It is important to wash the co-culture with C-solution to remove residual medium before treatment with vehicle or chemicals. Residual serum or phenol red from the culture medium may be a source of background fluorescence or an unwanted fluorescence quencher, respectively. They may thus interrupt the I-YFP-GJIC assay. Growth factors present in the FBS can also inhibit GJIC27. On the other hand, serum starvation can eventually lead apoptosis28, which can also be accelerated by chemical treatment. In our experience, the acceptor and donor cells were healthy after treatment by most screened chemicals at 25 &#181;M in C-solution without serum for 10 min. Some chemicals were not toxic even after treatment for 4 h. As chemical toxicity varies, the condition of the cells should be carefully checked when treatment for longer periods is required. If the cells are not healthy, an obvious reduction of YFP fluorescence that occurs within 1 s after introduction of the I-solution may result from cell detachment. That can be confirmed by microscopic observation of the involved well.
The cells used in the I-YFP-GJIC assay should meet several requirements. They should express GJs endogenously or be induced to form GJs. As channels composed of Cx43 are almost equally permeable to atomic cations and anions29, iodides can migrate through the channels present in I-YFP GJIC in LN215 cells. If the Cx channels in the cells of interest have low iodide permeability but freely allow diffusion of Ca2+, the I-YFP-GJIC assay may not be possible. The absence of iodide uptake activity is a requirement. The cells that express functional anion channels and uptake iodide rapidly are not appropriate for this assay. Cell growth is another requirement. The acceptor and donor cells are generated by transduction of lentiviruses expressing YFPQL or SLC26A4 and selection with puromycin for at least 1 week19. Cells that grow slowly or have limited growth such as primary hepatocytes are not suitable for this assay. Adhesion of cells to the surface of the plastic culture ware is also important. Cells that do not adhere firmly, such as HEK293 cells, are easily detached by introduction of the I-solution. This can be overcome by coating 96-well plates with PLL before plating. However, coating plates is a tedious step, and avoiding those types of cells is recommended. In principle, primary cells like astrocytes, hepatocytes, and keratinocytes are toxicologically, physiologically, and pharmacologically more relevant than cancer cells like glioma, hepatoma, and basal cell carcinoma cells. However, their limited growth rates make them inappropriate for the assay. Future advances in cell culture that allow growth of primary cells that meet the above requirements for longer periods, would broaden the toxicological, physiological, and pharmacological assessments possible with this assay.
The I-YFP GJIC assay can be used not only for HTS but also for determining whether a cell type endogenously expresses functional GJs. If the cells do not have GJs, the YFPQL quenching rate observed in cocultured acceptor and donor cells would not be different from that observed when only the acceptor cells were plated. If there was a significant difference in the quenching rates, then the original cells express functional GJs. As the difference tends to increase with time as shown in Figure 2A, this assay has a greater sensitivity to detect weak GJIC activity than scrape-loading or gap-FRAP assays. Time course data, dose-response relationships, and assessing the reversibility of GJ modulators can be obtained with the I-YFP-GJIC assay23,30. By inducing the expression of a Cx of interest into the cells devoid of functional GJs and then generating acceptors and donors, I-YFP specific Cx-GJIC assays can be established23. This assay thus makes it possible to determine IC50 values of a chemical on a specific type of Cx.
Like most HTS assays, the I-YFP-GJIC assay can produce false positive results resulting from changes in SLC26A4, YFPQL, or cellular iodide permeability. As false GJ inhibition can result from inhibition of SLC26A4 or desensitization of YFPQL to iodide, every potential GJ inhibitor should be tested using cells that coexpress YFPQL and SLC26A4. If a potential GJ inhibitor is an SLC26A4 blocker or a YFP desensitizer, YFP quenching is attenuated. On the contrary, a true GJ inhibitor does not affect YFP quenching. Electrophysiological methods or purified YFPQL can discriminate between SLC26A4 blockers and YFP desensitizers. GJ activators that sensitize YFP to iodide or increase iodide permeability via anion channels, Cx hemichannels, or by nonspecific toxic effects will also give false positive results. It should be determined whether potential GJ activators enhance YFP quenching when only acceptor cells are plated. If a chemical sensitizes YFPQL or increases iodide permeability, then it will increase YFPQL quenching in acceptor cell cultures without donors. The two types of false GJ activators can be distinguished in other GJ assays, such as scrape loading or FRAP or using purified YFPQL protein.

Gap junctions (GJs) act as intercellular channels to allow the diffusion of small molecules of &#60;1 kDa such as nutrients, metabolites, and signaling molecules between adjacent cells. The junctional elements include a hemichannel or connexon in each cell, and each connexon constitutes six connexins (Cxs)1. GJs and Cxs have been used in toxicology assays of carcinogens such as polycyclic aromatic hydrocarbons (PAH), which are GJ inhibitors2,3,4. Disrupted GJIC has been associated with nongenotoxic carcinogenesis5,6. As a potential therapeutic target, GJ involvement has been reported in particular subtypes of seizures7,8, protection from cardiac and brain ischemia/reperfusion injury9, migraine with aura10, drug-induced liver injury6,11, and wound healing12. High-throughput screening (HTS) assays are required to identify GJ-modulating chemicals or antibodies for drug discovery, for toxicology assays, and to identify novel cellular regulators of GJ activity. HTS assays can also be used to investigate structure-activity relationships of GJ modulators2,13,14,15.
Some GJIC assays include dye transfer or dual patch clamp techniques. Lucifer yellow CH (LY) and calcein acetoxymethyl ester (calcein-AM) have been used in dye-transfer assays. Cells are not permeable to LY, which is introduced by microinjection, scrape loading, or electroporation. Once inside the cell, LY spreads into neighboring cells via GJs and GJ activity is assayed by the extent of the LY migration16. Calcein-AM assays usually involve gap-fluorescence recovery after photobleaching17,18. Calcein-AM is a cell-permeant dye that is converted intracellularly into impermeable calcein by an intrinsic esterase. The assay requires a confocal microscope to observe the transfer of calcein-AM into a cell from those surrounding it following laser photobleaching. If functional GJs are present, calcein-AM in adjacent cells enters the photobleached cells and the fluorescence is recovered. GJ activity is assayed by the extent of fluorescence recovery of the photobleached cells. Dye-transfer assays are laborious and time consuming or have low sensitivities. Dual patch clamping is an electrophysiological method that measures junctional conductance. It is relatively sensitive, with a direct dependence of conductance on the number of open GJs19; however, it is technically demanding, time consuming, and expensive20. The I-YFP-GJIC assay was developed for use in HTS.
Figure 1 illustrates the components and steps of the I-YFP GJIC assay, which utilizes acceptor cells expressing an iodide-sensitive YFP variant bearing H148Q and I152L (YFPQL) and donor cells expressing an iodide transporter (SLC26A4)21. The two mutations carried by YFPQL allow quenching of fluorescence by iodide22. Iodides are added to co-cultured acceptor and donor cells; they do not enter the acceptor cells, but are taken up by the SLC26A4 transporters present on the donor cells. Iodides in the donor cells diffuse through functioning GJs into adjacent acceptor cells where they quench the YFPQL fluorescence. If GJs are closed or blocked by inhibitors, iodide cannot enter the acceptor cells to quench the fluorescence. The YFPQL quenching rate reflects GJ activity. The I-YFP GJIC assay procedure is neither complicated nor time consuming. It is compatible with HTS and can be used to test the effects of a large number of compounds on GJ activity in a relatively short period. It requires only acceptor and donor cells, and two balanced salt solutions. The protocol described below is based on LN215 cells whose major Cx is Cx4321. The LN215-YFPQL receptor and LN215-I&#8722; donor cells were generated by transduction with lentiviruses expressing YFPQL or SLC26A421,23.

<table><tbody><tr><td>96-well plate</td><td>SPL</td><td>30096</td><td></td></tr><tr><td>Calcium chloride (CaCl2)</td><td>Sigma</td><td>C5670</td><td>I-solution</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G7021</td><td>C-solution, I-solution</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>sigma</td><td>276855</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>RES6003H-B7</td><td>C-solution, I-solution</td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668-027</td><td>transfection reagent</td></tr><tr><td>Magnesium chloride hexahydrate (MgCl2 6H2O)</td><td>Sigma</td><td>M2393</td><td>C-solution</td></tr><tr><td>Microplate reader&amp;nbsp;</td><td>BMG LabTech&amp;nbsp;</td><td>POLARstar Omega 415-1618</td><td></td></tr><tr><td>pMD2.G&amp;nbsp;</td><td>Addgene</td><td>#12259</td><td></td></tr><tr><td>Polybrene</td><td>sigma</td><td>H9268</td><td></td></tr><tr><td>Poly-L-lysine solution</td><td>sigma</td><td>P4707</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma</td><td>P5405</td><td>C-solution, I-solution</td></tr><tr><td>psPAX2&amp;nbsp;</td><td>Addgene</td><td>&amp;nbsp;#12260</td><td></td></tr><tr><td>Puromycin Dihydrochloride</td><td>sigma</td><td>P8833</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma</td><td>S5886</td><td>C-solution, I-solution</td></tr><tr><td>Sodium hyroxide (NaOH)</td><td>Sigma</td><td>S2770</td><td></td></tr><tr><td>Sodium Iodide (NaI)</td><td>Sigma</td><td>383112</td><td>I-solution</td></tr></tbody></table>

an iodide-yellow fluorescent protein-gap junction-intercellular communication assay

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively. When iodide is added to a mixed culture of the two cell types, they enter the donor cells via the SLC26A4 transporter and diffuse to the adjacent acceptor cells via GJs where they quench the YFP fluorescence. YFP fluorescence is measured well by well in a kinetic mode. The YFP quenching rate reflects GJ activity. The assay is reliable and rapid enough to be used for HTS. The protocol for the I-YFP-GJIC assay using the LN215 cells, human glioma cells, is described.

Twenty-nine 96-well culture plates were used to screen 2,320 chemicals to identify novel GJIC modulators by I-YFP GJIC assay using the LN215-YFPQL and LN215-I&#8722; cells. The results obtained with a representative plate are shown in Figure 2. The percentage of YFP fluorescence in each well is shown as a line graph in Figure 2A and the percentage of GJIC activity in each well is shown in the bar graph in Figure 2B. The negative and positive controls and the 80 chemicals that were screened are shown in Table 1. Each well was treated with 25 &#181;M of a compound for 10 min. Terbinafine completely inhibited GJIC and homosalate inhibited approximately 50% of GJIC. Terbinafine was confirmed as a GJ inhibitor. Its dose-response, reversibility, and other experimental results have been published elsewhere23.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58966/58966fig1.jpg" /> 
Figure 1: The components and steps of the I-YFP GJIC assay (A) The yellow and dark yellow pentagons represent acceptor cells expressing YFPQL before and after quenching. The black pentagons are donor cells expressing SLC26A4. The blue bars are GJs and the red bars are SLC26A4 transporters. The pink circles are iodides. When the GJs are open (upper panel), iodides pass through SLC26A4 and enter the donor cell. Iodides migrate to adjacent acceptor cells via GJs. Iodides quench the YFP fluorescence of the acceptor cells. If the GJs are closed (lower panel), iodides entering the donor cells cannot move to the neighboring acceptor cells, and are retained only in the donor cells. The YFP fluorescence of the acceptor cells is not significantly reduced after the addition of iodides (B) Phase contrast and fluorescent images obtained when doing an I-YFP-GJIC assay. When acceptor and donor cells were plated at a ratio of 1:1, YFPQL quenching was observed 1 min after iodides were added (upper panel). When only acceptor cells were plated, iodide treatment for 1 min did not lead to significant YFPQL quenching21. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58966/58966fig1large.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/58966/58966fig2.jpg" /> 
Figure 2: Representative HTS results using the I-YFP-GJIC assay (A) Suspensions of 1:2 mixtures of LN215-YFPQL and LN215-I&#8722; cells were plated and incubated for 24 h, as described in protocol section 4. Cells were then washed and treated with vehicle or chemicals as in protocol section 5. All chemicals were assayed as 25 &#181;M samples from 2.5 mM stock solutions in DMSO for 10 min. Each well contained 1 &#181;L DMSO and 100 &#181;L of C-solution. The first and last rows of the plate were assigned to negative (vehicle) and positive (carbenoxolone) controls. The remaining 80 wells were used to screen chemicals listed in Table 1. After treatment, the I-YFP-GJIC assay was conducted well-by-well as in protocol section 5. The percentage of YFP fluorescence in each well at each time was normalized to the value at 2 s and plotted against time. The lines in the figure represent the changes in YFP fluorescence in each well. The YFP fluorescence in most well at 10 s ranged from 70% to 80%. Terbinafine and homosalate were potential hits for GJ inhibitors (B) The bar graph shows the percent GJIC activity of each of the 96 wells. The percent GJIC activity was calculated as shown in protocol step 6.1 and plotted in the bar graph against the well numbers. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58966/58966fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Well No.</td>
			<td>Position</td>
			<td>Chemical</td>
			<td>Well No.</td>
			<td>Position</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>1</td>
			<td>A01</td>
			<td>DMSO</td>
			<td>49</td>
			<td>E01</td>
			<td>DMSO</td>
		</tr>
		<tr>
			<td>2</td>
			<td>A02</td>
			<td>difloxacin hydrochloride</td>
			<td>50</td>
			<td>E02</td>
			<td>propofol</td>
		</tr>
		<tr>
			<td>3</td>
			<td>A03</td>
			<td>betamethasone valerate</td>
			<td>51</td>
			<td>E03</td>
			<td>oleandomycin phosphate</td>
		</tr>
		<tr>
			<td>4</td>
			<td>A04</td>
			<td>erythromycin</td>
			<td>52</td>
			<td>E04</td>
			<td>mianserin hydrochloride</td>
		</tr>
		<tr>
			<td>5</td>
			<td>A05</td>
			<td>cyproheptadine hydrochloride</td>
			<td>53</td>
			<td>E05</td>
			<td>valsartan</td>
		</tr>
		<tr>
			<td>6</td>
			<td>A06</td>
			<td>liothyronine</td>
			<td>54</td>
			<td>E06</td>
			<td>salsalate</td>
		</tr>
		<tr>
			<td>7</td>
			<td>A07</td>
			<td>theophylline</td>
			<td>55</td>
			<td>E07</td>
			<td>hydrocortisone</td>
		</tr>
		<tr>
			<td>8</td>
			<td>A08</td>
			<td>tolnaftate</td>
			<td>56</td>
			<td>E08</td>
			<td>rifaximin</td>
		</tr>
		<tr>
			<td>9</td>
			<td>A09</td>
			<td>trimethobenzamide hydrochloride</td>
			<td>57</td>
			<td>E09</td>
			<td>adrenolone hydrochloride</td>
		</tr>
		<tr>
			<td>10</td>
			<td>A10</td>
			<td>cefamandole nafate</td>
			<td>58</td>
			<td>E10</td>
			<td>imiquimod</td>
		</tr>
		<tr>
			<td>11</td>
			<td>A11</td>
			<td>dimethyl fumarate</td>
			<td>59</td>
			<td>E11</td>
			<td>nonoxynol-9</td>
		</tr>
		<tr>
			<td>12</td>
			<td>A12</td>
			<td>CBX</td>
			<td>60</td>
			<td>E12</td>
			<td>CBX</td>
		</tr>
		<tr>
			<td>13</td>
			<td>B01</td>
			<td>DMSO</td>
			<td>61</td>
			<td>F01</td>
			<td>DMSO</td>
		</tr>
		<tr>
			<td>14</td>
			<td>B02</td>
			<td>piracetam</td>
			<td>62</td>
			<td>F02</td>
			<td>ranolazine</td>
		</tr>
		<tr>
			<td>15</td>
			<td>B03</td>
			<td>gluconolactone</td>
			<td>63</td>
			<td>F03</td>
			<td>danthron</td>
		</tr>
		<tr>
			<td>16</td>
			<td>B04</td>
			<td>azlocillin sodium</td>
			<td>64</td>
			<td>F04</td>
			<td>acedapsone</td>
		</tr>
		<tr>
			<td>17</td>
			<td>B05</td>
			<td>choline chloride</td>
			<td>65</td>
			<td>F05</td>
			<td>atomoxetine hydrochloride</td>
		</tr>
		<tr>
			<td>18</td>
			<td>B06</td>
			<td>atorvastatin calcium</td>
			<td>66</td>
			<td>F06</td>
			<td>desoxycorticosterone acetate</td>
		</tr>
		<tr>
			<td>19</td>
			<td>B07</td>
			<td>oxyphencyclimine hydrochloride</td>
			<td>67</td>
			<td>F07</td>
			<td>tramadol hydrochloride</td>
		</tr>
		<tr>
			<td>20</td>
			<td>B08</td>
			<td>propafenone hydrochloride</td>
			<td>68</td>
			<td>F08</td>
			<td>terbinafine hydrochloride</td>
		</tr>
		<tr>
			<td>21</td>
			<td>B09</td>
			<td>fluconazole</td>
			<td>69</td>
			<td>F09</td>
			<td>topiramate</td>
		</tr>
		<tr>
			<td>22</td>
			<td>B10</td>
			<td>lovastatin</td>
			<td>70</td>
			<td>F10</td>
			<td>gemifloxacin mesylate</td>
		</tr>
		<tr>
			<td>23</td>
			<td>B11</td>
			<td>bleomycin (bleomycin b2 shown)</td>
			<td>71</td>
			<td>F11</td>
			<td>pravastatin sodium</td>
		</tr>
		<tr>
			<td>24</td>
			<td>B12</td>
			<td>CBX</td>
			<td>72</td>
			<td>F12</td>
			<td>CBX</td>
		</tr>
		<tr>
			<td>25</td>
			<td>C01</td>
			<td>DMSO</td>
			<td>73</td>
			<td>G01</td>
			<td>DMSO</td>
		</tr>
		<tr>
			<td>26</td>
			<td>C02</td>
			<td>acesulfame potassium</td>
			<td>74</td>
			<td>G02</td>
			<td>levalbuterol hydrochloride</td>
		</tr>
		<tr>
			<td>27</td>
			<td>C03</td>
			<td>teniposide</td>
			<td>75</td>
			<td>G03</td>
			<td>metformin hydrochloride</td>
		</tr>
		<tr>
			<td>28</td>
			<td>C04</td>
			<td>tannic acid</td>
			<td>76</td>
			<td>G04</td>
			<td>pregabalin</td>
		</tr>
		<tr>
			<td>29</td>
			<td>C05</td>
			<td>carprofen</td>
			<td>77</td>
			<td>G05</td>
			<td>topotecan hydrochloride</td>
		</tr>
		<tr>
			<td>30</td>
			<td>C06</td>
			<td>hydroxychloroquine sulfate</td>
			<td>78</td>
			<td>G06</td>
			<td>phenoxybenzamine hydrochloride</td>
		</tr>
		<tr>
			<td>31</td>
			<td>C07</td>
			<td>pentoxifylline</td>
			<td>79</td>
			<td>G07</td>
			<td>arecoline hydrobromide</td>
		</tr>
		<tr>
			<td>32</td>
			<td>C08</td>
			<td>mepivacaine hydrochloride</td>
			<td>80</td>
			<td>G08</td>
			<td>mepartricin</td>
		</tr>
		<tr>
			<td>33</td>
			<td>C09</td>
			<td>nilutamide</td>
			<td>81</td>
			<td>G09</td>
			<td>pantoprazole</td>
		</tr>
		<tr>
			<td>34</td>
			<td>C10</td>
			<td>aminolevulinic acid hydrochloride</td>
			<td>82</td>
			<td>G10</td>
			<td>loperamide hydrochloride</td>
		</tr>
		<tr>
			<td>35</td>
			<td>C11</td>
			<td>aniracetam</td>
			<td>83</td>
			<td>G11</td>
			<td>podofilox</td>
		</tr>
		<tr>
			<td>36</td>
			<td>C12</td>
			<td>CBX</td>
			<td>84</td>
			<td>G12</td>
			<td>CBX</td>
		</tr>
		<tr>
			<td>37</td>
			<td>D01</td>
			<td>DMSO</td>
			<td>85</td>
			<td>H01</td>
			<td>DMSO</td>
		</tr>
		<tr>
			<td>38</td>
			<td>D02</td>
			<td>metaxalone</td>
			<td>86</td>
			<td>H02</td>
			<td>levodopa</td>
		</tr>
		<tr>
			<td>39</td>
			<td>D03</td>
			<td>chloroguanide hydrochloride</td>
			<td>87</td>
			<td>H03</td>
			<td>ethisterone</td>
		</tr>
		<tr>
			<td>40</td>
			<td>D04</td>
			<td>clarithromycin</td>
			<td>88</td>
			<td>H04</td>
			<td>enrofloxacin</td>
		</tr>
		<tr>
			<td>41</td>
			<td>D05</td>
			<td>modaline sulfate</td>
			<td>89</td>
			<td>H05</td>
			<td>sparteine sulfate</td>
		</tr>
		<tr>
			<td>42</td>
			<td>D06</td>
			<td>protirelin</td>
			<td>90</td>
			<td>H06</td>
			<td>testosterone propionate</td>
		</tr>
		<tr>
			<td>43</td>
			<td>D07</td>
			<td>theobromine</td>
			<td>91</td>
			<td>H07</td>
			<td>pyridostigmine bromide</td>
		</tr>
		<tr>
			<td>44</td>
			<td>D08</td>
			<td>rosiglitazone maleate</td>
			<td>92</td>
			<td>H08</td>
			<td>enilconazole sulfate</td>
		</tr>
		<tr>
			<td>45</td>
			<td>D09</td>
			<td>losartan</td>
			<td>93</td>
			<td>H09</td>
			<td>betamethasone sodium phosphate</td>
		</tr>
		<tr>
			<td>46</td>
			<td>D10</td>
			<td>homosalate</td>
			<td>94</td>
			<td>H10</td>
			<td>azaserine</td>
		</tr>
		<tr>
			<td>47</td>
			<td>D11</td>
			<td>salicylanilide</td>
			<td>95</td>
			<td>H11</td>
			<td>acrisorcin</td>
		</tr>
		<tr>
			<td>48</td>
			<td>D12</td>
			<td>CBX</td>
			<td>96</td>
			<td>H12</td>
			<td>CBX</td>
		</tr>
	</tbody>
</table>
Table 1. List of chemicals screened in this study.

Watch this Scientific Journal Video about An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay at JoVE.com

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

Here, we present a protocol for a novel gap junction intercellular communication assay designed for the high-throughput screening of gap junction-modulating chemicals for drug discovery and toxicological assessment.

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and ...

an-iodide-yellow-fluorescent-protein-gap-junction-intercellular

Nanyang Technological University

Research

JoVE Journal

Biology

7.1K Views.  Yonsei University. Here, we present a protocol for a novel gap junction intercellular communication assay designed for the high-throughput screening of gap junction-modulating chemicals for drug discovery and toxicological assessment.

Video: An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

Cancer Research

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Bioengineering

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Recording Gap Junction Current from Xenopus Oocytes

Heterologous expression of connexins and innexins in Xenopus oocytes is a powerful approach for studying the biophysical properties of gap junctions (GJs). However, this approach is technically challenging because it requires a differential voltage clamp of two opposed oocytes sharing a common ground. Although a small number of labs have succeeded in performing this technique, essentially all of them have used either homemade amplifiers or commercial amplifiers that were designed for single-oocyte recordings. It is often challenging for other labs to implement this technique. Although a high side current measuring mode has been incorporated into a commercial amplifier for dual oocyte voltage-clamp recordings, there had been no report for its application until our recent study. We have made the high side current measuring approach more practical and convenient by introducing several technical modifications, including the construction of a magnetically based recording platform that allows precise placement of oocytes and various electrodes, use of the bath solution as a conductor in voltage differential electrodes, adoption of a commercial low-leakage KCl electrode as the reference electrode, fabrication of current and voltage electrodes from thin-wall glass capillaries, and positioning of all the electrodes using magnetically based devices. The method described here allows convenient and robust recordings of junctional current (Ij) between two opposed Xenopus oocytes.

Recording Gap Junction Current from Xenopus Oocytes

Here we present a protocol to express gap junction proteins in Xenopus oocytes and record junctional current between two apposed oocytes using a commercial amplifier designed for dual oocyte voltage-clamp recordings in a high side current measuring mode.

Heterologous expression of connexins and innexins in Xenopus oocytes is a powerful approach for studying the biophysical properties of gap junctions ...

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Bimolecular Fluorescence Complementation

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Single-cell Microinjection for Cell Communication Analysis

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

Recording Gap Junction Current from Xenopus Oocytes