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>

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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 exte...

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.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">96-well plate</td>
			<td style="width:72px;">SPL</td>
			<td style="width:123px;">30096</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Calcium chloride (CaCl2)</td>
			<td style="width:72px;">Sigma</td>
			<td style="width:123px;">C5670</td>
			<td>I-solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">D-(+)-Glucose</td>
			<td style="width:72px;">Sigma</td>
			<td style="width:123px;">G7021</td>
			<td>C-solution, I-solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:72px;">sigma</td>
			<td style="width:123px;">276855</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">HEPES</td>
			<td style="width:72px;">Sigma</td>
			<td style="width:123px;">RES6003H-B7</td>
			<td>C-solution, I-solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Lipofectamine 2000</td>
			<td style="width:72px;">Invitrogen</td>
			<td style="width:123px;">11668-027</td>
			<td>transfection reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">Magnesium chloride hexahydrate (MgCl2 6H2O)</td>
			<td style="width:72px;">Sigma</td>
			<td style="width:123px;">M2393</td>
			<td>C-solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">Microplate reader&#160;</td>
			<td style="width:72px;">BMG LabTech&#160;</td>
			<td style="width:123px;">POLARstar Omega 415-1618</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pMD2.G&#160;</td>
			<td style="width:72px;">Addgene</td>
			<td>#12259</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Polybrene</td>
			<td style="width:72px;">sigma</td>
			<td style="width:123px;">H9268</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Poly-L-lysine solution</td>
			<td style="width:72px;">sigma</td>
			<td style="width:123px;">P4707</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Potassium chloride (KCl)</td>
			<td style="width:72px;">Sigma</td>
			<td style="width:123px;">P5405</td>
			<td>C-solution, I-solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">psPAX2&#160;</td>
			<td style="width:72px;">Addgene</td>
			<td style="width:123px;">&#160;#12260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Puromycin Dihydrochloride</td>
			<td style="width:72px;">sigma</td>
			<td style="width:123px;">P8833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Sodium chloride (NaCl)</td>
			<td style="width:72px;">Sigma</td>
			<td style="width:123px;">S5886</td>
			<td>C-solution, I-solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Sodium hyroxide (NaOH)</td>
			<td style="width:72px;">Sigma</td>
			<td style="width:123px;">S2770</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Sodium Iodide (NaI)</td>
			<td style="width:72px;">Sigma</td>
			<td style="width:123px;">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 th...

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

Ein Jodid-gelb fluoreszierende Protein-Gap Junction-interzelluläre Kommunikation 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

Research

JoVE Journal

Biology

7.1K Aufrufe.  Yonsei University. Lückenkreuze wurden als Drogenziele vorgeschlagen. Der Iodide-YFP-GJIC-Assay ist mit einem Hochdurchsatz-Screening kompatibel, um Lückenknotenmodulatoren zu entdecken. Es wurde verwendet, um die Auswirkungen einer großen Anzahl von Verbindungen auf LückenknotenAktivitäten in relativ kurzer Zeit zu testen.Der Iodide-YFP-GJIC-Test ist einfach, robust, wiederholbar und kostengünstig. Dieser Test erfordert nur Akzeptor- und Spenderzellen und zwei ausgewogene Salzlösungen. Es werden ...

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

Titration of Human Coronaviruses Using an Immunoperoxidase Assay

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.

In this video, we demonstrate an alternative method for detection and titering of viruses using an enzymatic antigen detection technique known as an immunoperoxidase assay. Here, we will show you how to collect your viral samples, prepare the cells for testing, and finally the immunoperoxidase assay using serial dilutions to determine the viral titer.

Titration von Human Coronaviren Mit einem Immunoperoxidase Assay

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for ...

Forschung

Biologie

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Hausgemachte Mutagenese von Whole Plasmide

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

Ein In-vitro- FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

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.

Ein fluoreszierendes Screening Assay zur Identifizierung von Modulatoren der GIRK Kanäle

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 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.

Einzellige Mikroinjektions für Zellkommunikationsanalyse

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

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

Ein Test für Laterallinie Regeneration im erwachsenen Zebrafisch

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

Bewertung der Zebrafisch Nierenfunktion unter Verwendung einer fluoreszierenden Räumungs Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

Comet Assay als ein indirektes Maß für Systemische Oxidativer Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

Abbildung der molekularen Adhäsion im Zellrollen durch Adhäsion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can indirectly shape brain function. At the interface between the brain and the systemic circulation, the choroid plexuses (CP), which constitute the blood-cerebrospinal fluid barrier, have been highlighted as a key site of periphery-to-brain communication. CP produce the cerebrospinal fluid, neurotrophic factors, and signaling molecules that can shape brain homeostasis. CP are also an active immunological niche. In contrast to the brain parenchyma, which is populated mainly by microglia under physiological conditions, the heterogeneity of CP immune cells recapitulates the diversity found in other peripheral organs. The CP immune cell diversity and activity change with aging, stress, and disease and modulate the activity of the CP epithelium, thereby indirectly shaping brain function. The goal of this protocol is to isolate murine CP and identify about 90% of the main immune subsets that populate them. This method is a tool to characterize CP immune cells and understand their function in orchestrating periphery-to-brain communication. The proposed protocol may help decipher how CP immune cells indirectly modulate brain function in health and across various disease conditions.

This study uses flow cytometry and two different gating strategies on isolated perfused mice brain choroid plexuses; this protocol identifies the main immune cell subsets that populate this brain structure.

Isolierung und Charakterisierung der Immunzellen aus mikrodissektierten Maus-Aderhautplexus

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can ...