The authors would like to thank Herman Liggesmeyer and Marvin Bende for their expert advice in electrotechnics and informatics.

1. Assembly of a basic volt-amperemeter for TEER measurement
<ol>
	<li>Prepare a standard USB charger as the 5 V D.C. power supply, a USB extension cord, a microcontroller that will be used as a programmable square wave generator, two standard multimeters that are able to measure alternating current and voltage as root mean square (True-RMS), four cables with banana plugs, a telephone extension cord with a RJ14 female connector including six pins with the inner four wired (6P4C), two short cables, a luster terminal, a 120 k&#937; pre-resistor, wire end ferrules, and soldering lugs. The tools required are an insulation stripper, a crimping tool, and a soldering iron.</li>
	<li>First, connect the USB extension to the microcontroller board.</li>
	<li>Strip the end insulation of two short cables. Solder one side per cable either directly to pins 0 and 2 of the microcontroller or to soldering lugs, which in turn are clipped on the respective pins. Crimp the other ends to the wire end ferrules and connect them to a luster terminal as depicted in Figure 1.</li>
	<li>Link the banana plugs to the multimeters. Strip and crimp the other end of each of the four cables.</li>
	<li>Cut the telephone extension cord in two pieces and dismantle and crimp the conductors of the side containing the female connector. Check for the continuity of the conductors and pins.</li>
	<li>The first multimeter will be used to measure current in &#181;A (note that the AC mode has to be set explicitly). Connect it in a series with a 120 k&#937; pre-resistor to pins five and six of the RJ14 connector, corresponding to the outer electrode pair of the chopstick electrode.</li>
	<li>Finally, link the second multimeter, which will be used to measure the transepithelial voltage drop in mV, via the luster terminal to pins three and four of the RJ14 connector, corresponding to the inner electrode pair of the chopstick electrode.</li>
	<li>If desired, mount the installation in a chassis.</li>
</ol>
2. Programming the microcontroller
<ol>
	<li>Modify the provided source code (supplemental coding file 1) as needed. In the given form, pins 0 and 2 will alternate between ground and +5 V with 40 ms half-time of oscillation. Thus, a square wave signal with an amplitude of 5 V and a frequency of approximately 12.5 Hz will be generated. The real values may differ due to the inaccuracy of the microcontroller&#39;s time emitter.</li>
	<li>Connect the microcontroller to a desktop computer via a USB port and upload the source code with matching software25.</li>
</ol>
3. Recording of voltage oscillograms (optional)
<ol>
	<li>Bypass pins five and six of the RJ14 connector with a 1 k&#937; test resistor and connect to an oscilloscope.</li>
	<li>Check for the frequency, peak voltage, and waveform. Digitize and export the data.</li>
	<li>If desired, record oscillograms from a reference device (EVOM) and the self-assembled voltammeter for comparison. 
	NOTE: In this case, the data was recorded with a Digital Storage Scope HM 208. Being a very basic digital oscilloscope, the image could be internally digitized (frozen) but had to be plotted using an analogue PM 8143 X-Y recorder. The image was subsequently scanned.</li>
</ol>
4. Cell cultivation and TEER measurement
<ol>
	<li>Seed Human Choroid Plexus Papilloma (HIBCPP) cells on cell culture filter inserts with a pore size of 3 &#181;m in DMEM/F12 (see Table of Materials) containing 10% fetal calf serum9. Grow the cells at 37 &#176;C in a water saturated atmosphere containing 5% CO2 as described by Dinner et al.9.</li>
	<li>When the filters reach an impedance of 70 &#937; &#8729; cm2, change to serum-free DMEM/F12 and define the timepoint as Day 0.</li>
	<li>Connect the electrode to the RJ14 port of the self-assembled voltammeter and plug in the USB power supply. Set the multimeters to AC voltage mode (mV) and AC current mode (&#181;A), respectively.
	<ol>
		<li>Alternatively, connect the electrode to a commercially available reference device and turn on according to the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>Sterilize the electrode in 80% ethanol for 10 min and equilibrate in the appropriate medium for another 10 min.</li>
	<li>Put the electrode in both compartments of a cell culture filter insert system (the longer part of the electrode in the lower compartment and the shorter part in the upper compartment) containing a HIBCPP cell layer until the measurement values remain constant.</li>
	<li>For a reference device, note the impedance directly or calculate the impedance according to Ohm&#39;s law (R = U/I) for the self-assembled voltammeter. Be aware that electrode angle affects the measurements.</li>
	<li>Repeat the TEER measurement (steps 3&#8722;6) from Day 0 until Day 4.</li>
</ol>

<ol>
	<li>Matter, K., Balda, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+tight+junctions.">Functional analysis of tight junctions.</a> Methods. 30, 228-234 (2003).</li><li>Srinivasan, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TEER+measurement+techniques+for+in+vitro+barrier+model+systems.">TEER measurement techniques for in vitro barrier model systems.</a> Journal of Laboratory Automation. 20, 107-126 (2015).</li><li>Daniels, B. P., 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=Immortalized+human+cerebral+microvascular+endothelial+cells+maintain+the+properties+of+primary+cells+in+an+in+vitro+model+of+immune+migration+across+the+blood+brain+barrier.">Immortalized human cerebral microvascular endothelial cells maintain the properties of primary cells in an in vitro model of immune migration across the blood brain barrier.</a> Journal of Neuroscience Methods. 212, 173-179 (2013).</li><li>Weksler, B. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier-specific+properties+of+a+human+adult+brain+endothelial+cell+line.">Blood-brain barrier-specific properties of a human adult brain endothelial cell line.</a> Federation of American Societies for Experimental Biology Journal. 19, 1872-1874 (2005).</li><li>Lippmann, E. S., Al-Ahmad, A., Azarin, S. M., Palecek, S. P., Shusta, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+retinoic+acid-enhanced,+multicellular+human+blood-brain+barrier+model+derived+from+stem+cell+sources.">A retinoic acid-enhanced, multicellular human blood-brain barrier model derived from stem cell sources.</a> Scientific Reports. 4, 4160 (2014).</li><li>Stins, M. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+immortalized+human+cerebromicrovascular+endothelial+cell+line+as+an+in+vitro+model+of+the+human+blood-brain+barrier.">Development of immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood-brain barrier.</a> Federation of American Societies for Experimental Biology Journal. 11, 1187-1197 (1997).</li><li>Ishiwata, I., 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=Establishment+and+characterization+of+a+human+malignant+choroids+plexus+papilloma+cell+line+(HIBCPP).">Establishment and characterization of a human malignant choroids plexus papilloma cell line (HIBCPP).</a> Human Cell. 18, 67-72 (2005).</li><li>Dinner, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Choroid+Plexus+Epithelial+Cell-based+Model+of+the+Human+Blood-Cerebrospinal+Fluid+Barrier+to+Study+Bacterial+Infection+from+the+Basolateral+Side.">A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side.</a> Journal of Visualized Experiments. , (2016).</li><li>Schwerk, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+invasion+and+translocation+of+Neisseria+meningitidis+and+Streptococcus+suis+in+a+novel+human+model+of+the+blood-cerebrospinal+fluid+barrier.">Polar invasion and translocation of Neisseria meningitidis and Streptococcus suis in a novel human model of the blood-cerebrospinal fluid barrier.</a> PLoS One. 7, e30069 (2012).</li><li>Tenenbaum, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+bacterial+invasion+and+translocation+of+Streptococcus+suis+across+the+blood-cerebrospinal+fluid+barrier+in+vitro.">Polar bacterial invasion and translocation of Streptococcus suis across the blood-cerebrospinal fluid barrier in vitro.</a> Cellular Microbiology. 11, 323-336 (2009).</li><li>Gath, U., Hakvoort, A., Wegener, J., Decker, S., Galla, H. 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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+characteristics+of+calu-3+human+bronchial+epithelial+cells:+in+vitro-in+vivo+correlation+to+predict+lung+absorption+in+rats.">Permeability characteristics of calu-3 human bronchial epithelial cells: in vitro-in vivo correlation to predict lung absorption in rats.</a> Journal of Drug Targeting. 10, 31-40 (2002).</li><li>Fuchs, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+human+alveolar+epithelial+cells+in+primary+culture:+morphological+characterization+and+synthesis+of+caveolin-1+and+surfactant+protein-C.">Differentiation of human alveolar epithelial cells in primary culture: morphological characterization and synthesis of caveolin-1 and surfactant protein-C.</a> Cell and Tissue Research. 311, 31-45 (2003).</li><li>Furie, M. B., Cramer, E. B., Naprstek, B. L., Silverstein, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultured+endothelial+cell+monolayers+that+restrict+the+transendothelial+passage+of+macromolecules+and+electrical+current.">Cultured endothelial cell monolayers that restrict the transendothelial passage of macromolecules and electrical current.</a> The Journal of Cell Biology. 98, 1033-1041 (1984).</li><li>Hidalgo, I. J., Raub, T. J., Borchardt, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+human+colon+carcinoma+cell+line+(Caco-2)+as+a+model+system+for+intestinal+epithelial+permeability.">Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability.</a> Gastroenterology. 96, 736-749 (1989).</li><li>Yeste, 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=Geometric+correction+factor+for+transepithelial+electrical+resistance+measurements+in+Transwell+and+microfluidic+cell+cultures.">Geometric correction factor for transepithelial electrical resistance measurements in Transwell and microfluidic cell cultures.</a> Journal of Physics D Applied Physics. 49 (37), 3754 (2016).</li><li>Northrup, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VI:+The+Measurement+of+Low+Resistance.">VI: The Measurement of Low Resistance.</a> Methods of Measuring Electrical Resistance. , 100-131 (1912).</li><li>Li, H., Sheppard, D. N., Hug, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transepithelial+electrical+measurements+with+the+Ussing+chamber.">Transepithelial electrical measurements with the Ussing chamber.</a> Journal of Cystic Fibrosis. 3 (Suppl 2), 123-126 (2004).</li><li>Griep, 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=BBB+on+chip:+microfluidic+platform+to+mechanically+and+biochemically+modulate+blood-brain+barrier+function.">BBB on chip: microfluidic platform to mechanically and biochemically modulate blood-brain barrier function.</a> Biomedical Microdevices. 15, 145-150 (2013).</li><li>Esch, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+chip+porous+polymer+membranes+for+integration+of+gastrointestinal+tract+epithelium+with+microfluidic+'body-on-a-chip'+devices.">On chip porous polymer membranes for integration of gastrointestinal tract epithelium with microfluidic 'body-on-a-chip' devices.</a> Biomedical Microdevices. 14, 895-906 (2012).</li><li>. Arduino Web Editor Available from: <a target="_blank" href="https://www.arduino.cc/en/Main/Software">https://www.arduino.cc/en/Main/Software</a> (2019)</li><li>Benson, K., Cramer, S., Galla, 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=Impedance-based+cell+monitoring:+barrier+properties+and+beyond.">Impedance-based cell monitoring: barrier properties and beyond.</a> Fluids and Barriers of the CNS. 10, 5 (2013).</li><li>Hufnagl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Time Resolved Transepithelial Impedance Spectroscopy Of Caco 2 Monolayers Relying on Lithographically Patterned Basolateral Electrode Cell Arrays. , (2010).</li><li>Guimer&#224;, A., Gabriel, G., Parramon, D., Calder&#243;n, E., Villa, R., D&#246;ssel, O., Schlegel, W. 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The authors have no competing financial interests or other conflicts of interest.

Before a self-made voltammeter can be used in a daily routine, it is essential to check the device for proper function. In our case, a half-time of oscillation of 40 ms (12.5 Hz) was programmed, but the effective oscillation time turned out to be 60 ms (16.7 Hz). This inaccuracy of the microcontroller&#39;s time emitter had no detectable impact on TEER measurements. It might be best to determine the actual frequency using the frequency setting of one of the multimeters. If any deviation is found, the source code can be a...

Epithelial and endothelial cells function as cellular boundaries, separating the apical and basolateral sides of the body. If they are connected through tight junctions, passive substance diffusion through the paracellular spaces is restricted1, resulting in the formation of a selectively permeable barrier. Several artificial barrier systems have been developed2 using microvascular endothelial cells (HBMEC, blood-brain barrier3,4,5,6,7), choroid plexus epithelial cells (HIBCPP/PCPEC, blood-cerebrospinal fluid barrier8,9,10,11,12,13,14), colorectal adenocarcinoma cells (Caco-2, gastrointestinal models15), or airway/alveolar cell lines (pulmonary models16,17). These systems typically consist of cells grown in a monolayer on permeable membranes (i.e., filter insert systems) to allow access to the apical and basolateral sides. It is important that the integrity of the model system matches the in vivo conditions. Hence, several techniques have been developed to analyze barrier function by measuring paracellular diffusion of tracer compounds across the cell layer. These substances include radiolabeled sucrose, dye-labeled albumin, FITC-labeled inulin, or dye-labeled dextrans2. However, chemical dyes can make cells unusable for further experiments. To monitor barrier systems noninvasively, measurement of transepithelial/transendothelial electrical resistance (TEER) across a cellular monolayer can be used2,18,19. Because bipolar electrode systems are influenced by the electrode polarization impedance at the electrode-electrolyte interface, tetrapolar measurements are generally used to overcome this limitation20. The underlaying technique is a four-terminal sensing (4T) that was first described in 1861 by William Thomson (Lord Kelvin)21. In brief, the current is injected by a pair of current-carrying electrodes while a second pair of voltage-sensing electrodes is used to measure the voltage drop20. Nowadays, so-called chopstick electrodes consist of a pair of double electrodes, each containing a silver/silver-chloride pellet for measuring voltage and a silver electrode for passing current2. The electrical impedance is measured between the apical and the basolateral compartment with the cell layer in between (Figure 1). A square wave signal at a frequency of typically 12.5 Hz is applied at the outer electrodes and the resulting alternating current (AC) measured. Additionally, the potential drop across the cell layer is measured by the second (inner) electrode pair. Electrical impedance is then calculated according to Ohm&#39;s law. TEER values are normalized by multiplying impedance and cell layer surface area and are typically expressed as &#937; &#8729; cm2.
There are systems in which cells and electrodes are arranged in a more sophisticated way, but are also based on the 4T measuring principle and can be used with the same measurement devices. EndOhm systems, for example, in which the filter is inserted, contain a chamber and cap with a pair of concentric electrodes with the same structure as the chopstick electrode. The shape of the electrodes allows for a more uniform current density flow across the membrane, thereby reducing variation between readings. Even more complex (but also more accurate) is an Ussing chamber, where a cell layer separates two chambers filled with Ringer&#39;s solution22. The chamber itself can be gassed with oxygen, CO2, or N2, and stirred or supplemented with experimental substances. As ion transport across the cell layer occurs, a potential difference can be measured by two voltage-sensing electrodes near the tissue. This voltage is cancelled out by two current-carrying electrodes placed next to the cell layer. The measured current will then give the net ion transport and the transepithelial resistance, which reflects barrier integrity, can be determined22. TEER measurement can also be applied on body-on-a-chip systems that represent barrier-tissue models23,24. These systems mimic in vivo conditions of the cells and often consist of several types of cells, stacked on top of each other in layers.
The following protocol explains how to set up a cost-effective and reliable voltammeter with programmable output frequency that produces no statistically significant differences in TEER compared to commercially available measurement systems.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>120 kOhm resistor</td>
			<td></td>
			<td></td>
			<td>General (generic) equipment</td>
		</tr>
		<tr>
			<td>Banana plug cables</td>
			<td></td>
			<td></td>
			<td>General (generic) equipment</td>
		</tr>
		<tr>
			<td>Cables</td>
			<td></td>
			<td></td>
			<td>General (generic) equipment</td>
		</tr>
		<tr>
			<td>Chopstick electrode</td>
			<td>Merck Millicell</td>
			<td>MERSSTX01</td>
			<td></td>
		</tr>
		<tr>
			<td>Chopstick electrode (alternative)</td>
			<td>WPI World Precision Instruments</td>
			<td>STX2</td>
			<td></td>
		</tr>
		<tr>
			<td>Crimping tool</td>
			<td></td>
			<td></td>
			<td>General tool</td>
		</tr>
		<tr>
			<td>Digispark / ATtiny85</td>
			<td>AZ-Delivery Vertriebs GmbH</td>
			<td>Digispark Rev.3 Kickstarter</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM:F12</td>
			<td>Gibco (Thermo Fisher)</td>
			<td>31330038</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)/Fetal Bovine Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter inserts 3&#181;m translucent</td>
			<td>Greiner Bioone</td>
			<td>662631</td>
			<td></td>
		</tr>
		<tr>
			<td>HIBCPP</td>
			<td></td>
			<td></td>
			<td>Hiroshi Ishikawa / Horst Schroten</td>
		</tr>
		<tr>
			<td>Insulation stripper</td>
			<td></td>
			<td></td>
			<td>General tool</td>
		</tr>
		<tr>
			<td>Luster terminal</td>
			<td></td>
			<td></td>
			<td>General (generic) equipment</td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>HAMEG</td>
			<td>Digital Storage Scope HM 208</td>
			<td></td>
		</tr>
		<tr>
			<td>Plotter</td>
			<td>PHILIPS</td>
			<td>PM 8143 X-Y recorder</td>
			<td></td>
		</tr>
		<tr>
			<td>Software Arduino</td>
			<td>https://www.arduino.cc</td>
			<td>Arduino 1.8.9</td>
			<td></td>
		</tr>
		<tr>
			<td>Soldering iron</td>
			<td></td>
			<td></td>
			<td>General tool</td>
		</tr>
		<tr>
			<td>Soldering lugs</td>
			<td></td>
			<td></td>
			<td>General (generic) equipment</td>
		</tr>
		<tr>
			<td>Telephone cable with RJ14 (6P4C) connector</td>
			<td></td>
			<td></td>
			<td>General (generic) equipment</td>
		</tr>
		<tr>
			<td>Test resistor</td>
			<td>Merck Millicell</td>
			<td>MERSSTX04</td>
			<td></td>
		</tr>
		<tr>
			<td>True-RMS multimeters</td>
			<td>VOLTCRAFT</td>
			<td>VC185</td>
			<td></td>
		</tr>
		<tr>
			<td>USB charger</td>
			<td></td>
			<td></td>
			<td>General (generic) equipment</td>
		</tr>
		<tr>
			<td>USB extension cord</td>
			<td></td>
			<td></td>
			<td>General (generic) equipment</td>
		</tr>
		<tr>
			<td>Voltohmmeter for TEER measurement</td>
			<td>WPI World Precision Instruments</td>
			<td>EVOM</td>
			<td></td>
		</tr>
		<tr>
			<td>Voltohmmeter for TEER measurement (alternative)</td>
			<td>Merck Millicell</td>
			<td>ERS</td>
			<td></td>
		</tr>
		<tr>
			<td>Wire end ferrules</td>
			<td></td>
			<td></td>
			<td>General (generic) equipment</td>
		</tr>
	</tbody>
</table>

a simple approach to perform teer measurements using a self-made volt-amperemeter with programmable output frequency

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model systems. In most cases, chopstick electrodes are used to determine the electric impedance between the upper and lower compartment of a cell culture filter insert system containing cellular monolayers. The filter membrane allows the cells to adhere, polarize, and interact by building tight junctions. This technique has been described with a variety of different cell lines (e.g., cells of the blood-brain barrier, blood-cerebrospinal fluid barrier, or gastrointestinal and pulmonary tract). TEER measurement devices can be readily obtained from different laboratory equipment suppliers. However, there are more cost-effective and customizable solutions imaginable if an appropriate voltammeter is self-assembled. The overall aim of this publication is to set up a reliable device with programmable output frequency that can be used with commercially available chopstick electrodes for TEER measurement.

To compare the operation of a self-assembled voltammeter with its commercially available counterpart, a voltage oscillogram of both devices was recorded.
As shown in Figure 2A, the reference instrument generated a square wave signal with an amplitude of 80 mV and an oscillation time of 80 ms, which corresponds to a frequency of 12.5 Hz, when operating on-load with a 1 k&#937; test resistor.
...

Watch this Scientific Journal Video about A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency at JoVE.com

A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency

Here, we demonstrate how to set up an inexpensive volt-amperemeter with programmable output frequency that can be used with commercially available chopstick electrodes for transepithelial/endothelial electrical resistance measurements.

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model ...

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21.4K 조회수.  University of Tübingen. Here, we demonstrate how to set up an inexpensive volt-amperemeter with programmable output frequency that can be used with commercially available chopstick electrodes for transepithelial/endothelial electrical resistance measurements.

비디오: A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

Microfabricated 포스트 - 배열 감지기 (mPADs) : 기계적 힘을 분리하는 접근

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

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생물학

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades. Today, such pipettes are made from glass capillaries using a programmable puller. Such instruments heat the capillary using either a metal filament or a laser and draw out the glass using gravity, a motor or both. Pipettes for patch-clamp recording are formed using only heat and gravity, while sharp electrodes for intracellular recording use a combination of heat, gravity, and a motor. The procedure used to make intracellular recording pipettes is similar to that used to make injection needles for a variety of applications, including cRNA injection into Xenopus oocytes. In general, capillary glass &lt;1.2 mm in diameter is used to make pipettes for patch clamp recording, while narrower glass is used for intracellular recording (outer diameter = 1.0 mm). For each tool, the puller is programmed slightly differently. This video shows how to make both kinds of recording pipettes using pre-established puller programs.

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

프로그래밍 풀러와 패치 - pipettes와 샤프 전극을 만들기

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades.  Today, such pipettes are made from glass capillaries ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

고효율 기본 셀을 Transfect하는 진 Pulser Electroporation MXcell의 시스템을 사용하여

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

Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are exciting cell sources due to their limitless self-renewal capabilities and their potential to differentiate into multiple cell types. The pluripotent state of hPSCs is typically assessed by techniques such as qPCR, immunocytochemistry, and by other in vitro and in vivo differentiation strategies into multiple cell types. Among these, immunocytochemical techniques have been developed for routine characterization of the undifferentiated state of hPSCs based on analysis of candidate intracellular and cell-surface biomarkers. Given the fact that hPSCs grow as colonies, problems arise in quantifying the expression of these markers at the individual cell level on a routine basis. Flow cytometry analyses serve to address this issue but require cell numbers and use of reagents that are not normally conducive for routine quality control assessment of hPSC cultures. Thus, the development of practical and reproducible means of creating monolayer cell samples with preserved integrity for marker evaluation has many advantages in stem cell research. This greatly benefits immunocytochemical analysis because individual cells from the monolayer can be easily observed and quantified for the expression of specific markers. Towards this goal, a self-made cytospin apparatus was constructed and optimized for use with immunocytochemical staining. Two cell-surface markers (SSEA3/SSEA4) expression were analyzed in a variant BG01 stem cell line for the purpose of this protocol.

Suspension immunocytochemical staining of human pluripotent stem cells (hPSCs) for cell-surface markers (SSEA-3/SSEA-4) was achieved based on use of a self-made cytospin apparatus to create a monolayer of cells for observation and quantification.

자수 성 가한 Cytospin 장치를 사용하여 인간 Pluripotent 줄기 세포의 Immunocytochemical 분석

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are exciting cell ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Embryogenesis 동안 중복 기능을 테스트하기 위해 역방향 유전자 접근

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability

The ability to modify/visualize biological surfaces, and then study the modified cell/virion in a range of in vitro and in vivo environments is essential to gaining further insight into the function of specific molecules or the entire entity. Studies of biological surface modification are generally limited to genetic engineering of the organism or the covalent attachment of chemical moieties to the cell surface1,2. However these traditional techniques expose the cell to chemical reactants, or they require significant manipulation to achieve the desired outcome, making them cumbersome, and they may also inadvertently affect the viability/functionality of the modified cell. A simple method to harmlessly modify the surface of cells is required.
Recently a new technology, KODE Technology has introduced a range of novel constructs consisting of three components: a functional head group (F), a spacer (S) and a lipid tail (L) and are known as Function-Spacer-Lipid or FSL constructs3. The spacer (S) is selected to provide a construct that is dispersible in water, yet will spontaneously and stably incorporate into a membrane. 
FSL construct functional moieties (F) so far include a range of saccharides including blood group-related determinants, sialic acids, hyaluronan polysaccharides, fluorophores, biotin, radiolabels, and a range of peptides3-12. FSL constructs have been used in modifying embryos, spermatozoa, zebrafish, epithelial/endometrial cells, red blood cells, and virions to create quality controls systems and diagnostic panels, to modify cell adhesion/ interaction/ separation/ immobilization, and for in vitro and in vivo imaging of cells/virions3-12.
The process of modifying cells/virions is generic and extremely simple. The most common procedure is incubation of cells (in lipid free media) with a solution for FSL constructs for 1-2 hours at 37&deg;C4-10. During the incubation the FSL constructs spontaneously incorporate into the membrane, and the process is complete. Washing is optional. Cells modified by FSL constructs are known as kodecytes6-9, while virions are kodevirions10.
FSL constructs as direct infusions and kodecytes/kodevirions have been used in experimental animal models7,8,10. All kodecytes/kodevirions appear to retain their normal vitality and functionality while gaining the new function of the F moiety7,8,10,11.
The combination of dispersibility in biocompatible media, spontaneous incorporation into cell membranes, and apparent low toxicity, makes FSL constructs valuable research tools for the study of cells and virions.

Function-Spacer-Lipid (FSL) constructs allow the surface characteristics of living cells and virions to be modified without loss of vitality. The method requires only simple contact of an FSL construct solution with a cell/virion and spontaneous and stable surface incorporation occurs.

FSL은 구조 : 생물 학적 마커의 범위와 셀 / Virion 표면을 수정하기위한 간단한 방법을 자신의 생존에 영향을주지 않고

The ability to modify/visualize biological surfaces, and then study the modified cell/virion in a range of in vitro and in vivo environments is essential ...

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8.
The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization.
In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

주름 식민지 개발을 사용하여 Biofilm 형성을 평가하기 위해 세미 양적 접근

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a ...

Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro positive selection assay to measure in vivo mutations induced in a transgenic &#955;gt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.

De novo mutations in the male germline may contribute to adverse health outcomes in subsequent generations. Here we describe a protocol for the use of a transgenic rodent model for quantifying mutations in male germ cells induced by environmental agents.

형질 전환 쥐 분석 정량화 남성 생식 세포 돌연변이 주파수에 대한

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for ...

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

핵심 생물학 개념을 가르치는 마우스 유방 종양 세포를 사용 : 간단한 랩 모듈

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

형광 현미경으로 Stromule 주파수를 시각화

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...