This work was supported by the National Institutes of Health grant number R21-HL094828-01 awarded to Dr. Poelzing.

1. Prepare Noise Function and Input Signal
<ol>
<li>Create a noise function containing the desired frequency components. This can be done by describing the desired frequency components in the frequency domain and then calculating the inverse fast Fourier transform. In this study, 1 - 15 kHz was used. All Fourier transforms and inverse Fourier transforms described in this study were calculated using Matlab's FFT and IFFT functions.</li>
<li>Scale the amplitude of the noise function appropriately. In this study the noise function was scaled such that the zero to peak amplitude of the noise function was 50 or 100 mV.</li>
<li>Create a stimulus file using methods appropriate for the acquisition software being used. For Clampex 8, first create a text file with the appropriate header. Below the header, insert the time increments for a single sweep in the first column. The time increments should have the same temporal spacing as the sampling interval used in the measurements. For each sweep in the voltage step protocol insert the exact voltages desired at each time step. This should include the noise function.</li>
</ol>
2. Perform Voltage Clamp Measurements
<ol>
<li>Create a measurement protocol within the acquisition software that is compatible with the stimulus file generated previously. In Clampex, there is a menu that allows the user to associate a stimulus file with the current protocol.</li>
<li>Attach a model (or biological) cell to the measurement equipment.</li>
<li>Perform the experiment as scheduled. For control purposes, make sure to include periodic measurements that do not include any noise functions.</li>
</ol>
3. Post Experiment Analysis
<ol>
<li>Calculate the IV curve for an individual recording. If the recording is at steady state when the noise function is applied, the IV curve can be created using a steady state portion of the recording temporally outside the range of the noise function. If the recording was not at steady state, the noise function may interfere with calculation of the IV curve, so a second recording should be made without the noise function present. </li>
<li>For each voltage step in a recording calculate the fast Fourier transform of the portion of the recording where the noise function was inserted. Combine the Fourier transform for each voltage step into a m x n matrix, where m is the number of frequencies in the FFT, and n is the number of voltage steps. In this configuration, each row of the matrix represents the amplitude of a single frequency at all voltage steps in the experiment.</li>
<li>For each frequency (ie each row in the above matrix) correlate the row with the IV curve generated in 3.1 and record the correlation coefficient.</li>
<li>Plot the correlation coefficient vs frequency to visualize frequencies which highly correlate with the IV curve. Since the DC component is contained within the first frequency of the FFT, the correlation coefficient for this frequency should always be &gt; 0.99. </li>
</ol>
4. Representative Results:

Representative voltage clamp measurements are shown for a model cell without (Figure 1A) and with (Figure 1B) a noise function inserted into the voltage step protocol. The IV curve was also calculated for the model cell (Figure 1C). For each sweep, in the recordings from Figure 1, the FFT was calculated over the time frame where the noise function was inserted (see red box in Figures 1A, 1B). Figures 2A and 2B show the FFTs calculated for the recordings shown in Figure 1A and 1B, respectively. Upon visual inspection, the DC component (highlighted in red) appears to mimic the shape of the IV curve. Without the noise function, all frequencies above DC appear to have amplitudes near zero (Figure 2A). When the noise function is inserted, frequencies between 1 and 15 kHz have visually noticeable amplitudes (Figure 2B). Figure 3 shows the result of correlating individual frequency amplitudes over the range of voltage steps against the IV curve. Figure 3A-C shows the correlation coefficients when the experiment was done under control conditions (no noise function) and with noise amplitudes of 50 and 100 mV, respectively. Notice in all cases, the DC component appears to correlate nearly perfectly with the IV curve. Indeed, for all recordings, the correlation coefficient for this frequency was greater than 0.99 (R = 0.9996 &plusmn; 1E-5, mean &plusmn; standard deviation). When we look at Figure 3A (control conditions), there are no frequencies besides the DC component whose amplitude significantly correlates with the IV curve. Specifically, none of these frequencies have correlation coefficients greater than 0.5. Upon insertion of the lowest amplitude noise function (50 mV), these same frequencies still had correlation coefficients less than 0.5. The correlation coefficients for these frequencies also did not become greater than 0.5 when the noise amplitude was increased to 100 mV.

 <img src="/files/ftp_upload/2361/2361fig1.jpg" alt="Figure 1" /> 
Figure 1. Model Cell Recordings: Voltage clamp recordings are shown for a model cell without (1A) and with (1B) a noise function included in the voltage step protocol. For the first and last 20 ms of each sweep, the potential was kept at the holding potential (0 mV). Each voltage step was 80 ms long, and the noise function was inserted 40 ms after the beginning of the step. The noise function had a duration of 30 ms and contained frequencies between 1 and 15 kHz. Voltage was stepped fro, -150 to +50 mV in 5 mV increments. An IV curve for the model cell is also shown (1C). To make the recordings easier to read, only every fifth sweep was included in 1A and 1B, but all sweeps were included in 1C.
 <img src="/files/ftp_upload/2361/2361fig2.jpg" alt="Figure 2" /> 
Figure 2. FFT of Recordings: The FFT was calculated for the 30 ms portion of each sweep where the noise function is to be inserted (the area bounded by the red box in Figures 1A, 1B). Figures 2A and 2B show the FFTs calculated without and with the noise function, respectively. Once again, for clarity, only the FFT from every fifth sweep is included in the figure, but all sweeps were used in future calculations.
 <img src="/files/ftp_upload/2361/2361fig3.jpg" alt="Figure 3" /> 
Figure 3. IV Frequency Correlations: The results of correlations between the IV curve of a recording and the amplitude of individual frequencies over the range of voltage steps is shown. Figures 1A - 1C show the correlation coefficient for frequencies from 0 to 20 kHz under control conditions, and in the presence of 50 or 100 mV noise functions, respectively.
 <img src="/files/ftp_upload/2361/2361fig4.jpg" alt="Figure 4" /> 
Figure 4. Re-Creation of the IV Curve: The IV curve for the model cell (same as Figure 1C) and the magnitude of two frequencies were overlaid. The first frequency was the DC component (R = 0.995), and the second was a randomly chosen frequency with low correlation (R = 0.3212). The frequency amplitudes were scaled to approximately the same amplitude as the IV curve.

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	<li>Hamill, O. P., Marty, A., Neher, E., Sakmann, B., Sigworth, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+patch-clamp+techniques+for+high-resolution+current+recording+from+cells+and+cell-free+membrane+patches.">Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.</a> Pflugers Arch. 391, 85-100 (1981).</li><li>Ukomadu, C., Zhou, J., Sigworth, F. J., Agnew, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mu]l+Na++channels+expressed+transiently+in+human+embryonic+kidney+cells:+Biochemical+and+biophysical+properties.">mu]l Na+ channels expressed transiently in human embryonic kidney cells: Biochemical and biophysical properties.</a> Neuron. 8, 663-676 (1992).</li><li>Abriel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Arrhythmogenic+Mechanism+Revealed+by+a+Long-QT+Syndrome+Mutation+in+the+Cardiac+Na++Channel.">Novel Arrhythmogenic Mechanism Revealed by a Long-QT Syndrome Mutation in the Cardiac Na+ Channel.</a> Circ Res. 88, 740-745 (2001).</li><li>Giles, W. R., Imaizumi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+potassium+currents+in+rabbit+atrial+and+ventricular+cells.">Comparison of potassium currents in rabbit atrial and ventricular cells.</a> J Physiol. 405, 123-145 (1988).</li><li>Lee, K. S., Tsien, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+calcium+channel+blockade+by+verapamil,+D600,+diltiazem+and+nitrendipine+in+single+dialysed+heart+cells.">Mechanism of calcium channel blockade by verapamil, D600, diltiazem and nitrendipine in single dialysed heart cells.</a> Nature. 302, 790-794 (1983).</li><li>Ozdemir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+Inhibition+of+Na/Ca+Exchange+Results+in+Increased+Cellular+Ca2++Load+Attributable+to+the+Predominance+of+Forward+Mode+Block.">Pharmacological Inhibition of Na/Ca Exchange Results in Increased Cellular Ca2+ Load Attributable to the Predominance of Forward Mode Block.</a> Circ Res. 102, 1398-1405 (2008).</li><li>Zhang, S., Zhou, Z., Gong, Q., Makielski, J. C., January, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+Block+and+Identification+of+the+Verapamil+Binding+Domain+to+HERG+Potassium+Channels.">Mechanism of Block and Identification of the Verapamil Binding Domain to HERG Potassium Channels.</a> Circ Res. 84, 989-998 (1999).</li><li>Veeraraghavan, R., Poelzing, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+underlying+increased+right+ventricular+conduction+sensitivity+to+flecainide+challenge.">Mechanisms underlying increased right ventricular conduction sensitivity to flecainide challenge.</a> Cardiovasc. Res. 77, 749-756 (2008).</li><li>Goodman, M., Art, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+feedback+by+a+potassium-selective+inward+rectifier+enhances+tuning+in+vertebrate+hair+cells.">Positive feedback by a potassium-selective inward rectifier enhances tuning in vertebrate hair cells.</a> Biophysical Journal. 71, 430-442 (1996).</li><li>Han, A., Frazier, A. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+description+of+three+modes+of+activity+of+fast+chloride+channels+from+rat+skeletal+muscle.">Quantitative description of three modes of activity of fast chloride channels from rat skeletal muscle.</a> J Physiol. 378, 141-174 (1986).</li><li>Kuo, J. J., Lee, R. H., Zhang, L., Heckman, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+of+the+persistent+sodium+current+in+spike+initiation+during+slowly+rising+inputs+in+mouse+spinal+neurones.">Essential role of the persistent sodium current in spike initiation during slowly rising inputs in mouse spinal neurones.</a> The Journal of Physiology. 574, 819-834 (2006).</li><li>Thompson, A. 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No conflicts of interest declared.

There are technical obstacles that presently prevent researchers from measuring multiple ion channel types simultaneously with the intent of later determining how much current should be attributed to each channel type. Because of this limitation, ion channels are usually studied individually using techniques such as voltage, current, and action potential clamp.1 To study individual channel types, heterologous expression systems are often used.2 When working with cells isolated from tissue, such as cardiomyocytes, other means must be used to block various ion channels. For example, sodium channels can be inactivated by a slow depolarizing voltage ramp,3 inward rectifying potassium channels can be blocked with extracellular BaCl2,4 and calcium channels can be blocked using verapamil.5
One method used that partially overcomes this limitation is to measure current flux through two channels types simultaneously, then repeat the measurement after selectively blocking one channel type with an appropriate agent. Subtraction of the two measurements can then be used to estimate the amount of current attributable to the channel type that was blocked.6 However, there are two major limitations to this technique. First, chemical agents have not been identified that can selectively block each ion channel, and some widely used drugs have non-specific interactions with other channel types.5,7 Second, it cannot be determined from this technique whether one channel is modulated by another channel. For example, heterogeneous expression of NaV 1.5 and Kir 2.1 has been shown in guinea pig ventricles, and it has been suggested that a synergistic relationship exists between the two channels, such that higher Kir 2.1 expression in the right ventricle depresses conduction velocity.8 Presently, this cannot be verified.
In this study, we suggest that impedance spectroscopy may be a useful tool for studying multiple ion channel types measured simultaneously. Although the method presented has never been used to discern currents from two channel types measured simultaneously, impedance spectroscopy has been used to study a number of other aspects of ion channel function. Goodman and Art showed using turtle auditory hair cells that current clamp protocols can be modified to tune a cell to different frequencies, and the oscillations in the transmembrane potential is due to an interplay between an inward rectifying K+ channel and a Ca2+ channel.9 Han and Frazier demonstrated that impedance can be measured in a single cell over a wide range of frequencies ( 100 hZ to 5 MhZ), and the increase in impedance observed when K+ or Ca2+ channels were blocked could be a simple means to detect channel block in high throughput drug screens.10 Hayashi and Fishman have used complex conductance to study kinetic properties of an inward rectifying K+ channel.11 Other groups have inserted a single frequency into the voltage clamp protocol of different channel types and showed that the observed frequency response agreed with the expected response for some frequencies but not others.12,13 Millonas and Hanck suggested the reason some frequencies did not produce the expected response is the presence of multiple rate constants in the Markov model.12 Studies such as these, as well as others, have demonstrated that there are instances when ionic currents measured from ion channels while using impedance spectroscopy do not agree with the theoretical frequency response. This is not a concern in this study because the purpose of the method in this study is to identify frequencies that correlate with the current amplitude independent of the underlying assumptions of the membrane electrical circuit. Furtheremore, the current amplitude is calculated from portions of the recordings that do not have any noise functions inserted into them. A number of other studies also present models of numerous ion channels exhibiting numerous conducting and non-conduction states all with their own rate constants.14,15,16 Thompson et al showed that the selectivity filter of the KcsA channel has different binding sites for Na+, Li+, and K+, and the energetic costs of moving from one binding site to another as an ion moves through the selectivity filter is what makes the channel preferentially conduct K+ ions through its pore.17 In this paper we inserted a range of frequencies (noise function) into a voltage step protocol and looked for frequencies whose amplitude highly correlate with the overall current amplitude. Since strong evidence has been presented suggesting multiple rate constants play a role in conduction of ions through different channels, introduction of the frequencies associated with these rate constants may cause certain frequencies to resonate or highly correlate with the current amplitude, which would not have otherwise. The technique demonstrated in this study is performed on a model cell, which is a parallel RC circuit that is usually used to test the voltage clamp circuitry and acquisition equipment. It is not expected that any frequencies besides DC would correlate with the current magnitude, and this is shown in our data. We also show that addition of the noise function did not cause any frequencies to highly correlate with the current amplitude. These two findings are critical because they show that the measurement equipment and noise function do not by themselves cause any frequencies to correlate with the current amplitude. When future studies make measurements using membranes containing ion channels, it is expected that, depending on the channel used, frequencies that correspond to rate constants in the selectivity filter or possibly the pore will influence the frequency response of the channel and affect which frequencies have high or low correlation with the current amplitude.
Since this method is a new technique for studying ion channels, there are a number of directions future studies could follow. First, the technique should be used to characterize the frequency response of specific isolated channels. Additional work also needs to be done to calibrate the frequency amplitudes to the current amplitudes. Once multiple channels are characterized individually, multiple channel types should be measured simultaneously. The technique could also be adapted for use in action potential clamp, current clamp, and field stimulation studies. While this is a new technique, it shows what may be a powerful way to make electrophysiological measurements that were previously not possible and provide valuable new insights into the physiological role of ion channels.

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<td>Matlab</td>
<td>&nbsp;</td>
<td>Mathworks</td>
<td>n/a</td>
<td>Natick, MA</td>
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<td>Clampex 8</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>Clampex 8</td>
<td>Sunnyvale, CA</td>
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<td>Integrating Patch Clamp Amplifier</td>
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recapitulation of an ion channel iv curve using frequency components

INTRODUCTION: Presently, there are no established methods to measure multiple ion channel types simultaneously and decompose the measured current into portions attributable to each channel type. This study demonstrates how impedance spectroscopy may be used to identify specific frequencies that highly correlate with the steady state current amplitude measured during voltage clamp experiments. The method involves inserting a noise function containing specific frequencies into the voltage step protocol. In the work presented, a model cell is used to demonstrate that no high correlations are introduced by the voltage clamp circuitry, and also that the noise function itself does not introduce any high correlations when no ion channels are present. This validation is necessary before the technique can be applied to preparations containing ion channels. The purpose of the protocol presented is to demonstrate how to characterize the frequency response of a single ion channel type to a noise function. Once specific frequencies have been identified in an individual channel type, they can be used to reproduce the steady state current voltage (IV) curve. Frequencies that highly correlate with one channel type and minimally correlate with other channel types may then be used to estimate the current contribution of multiple channel types measured simultaneously.
METHODS: Voltage clamp measurements were performed on a model cell using a standard voltage step protocol (-150 to +50 mV, 5mV steps). Noise functions containing equal magnitudes of 1-15 kHz frequencies (zero to peak amplitudes: 50 or 100mV) were inserted into each voltage step. The real component of the Fast Fourier transform (FFT) of the output signal was calculated with and without noise for each step potential. The magnitude of each frequency as a function of voltage step was correlated with the current amplitude at the corresponding voltages.
RESULTS AND CONCLUSIONS: In the absence of noise (control), magnitudes of all frequencies except the DC component correlated poorly (|R|&lt;0.5) with the IV curve, whereas the DC component had a correlation coefficient greater than 0.999 in all measurements. The quality of correlation between individual frequencies and the IV curve did not change when a noise function was added to the voltage step protocol. Likewise, increasing the amplitude of the noise function also did not increase the correlation. Control measurements demonstrate that the voltage clamp circuitry by itself does not cause any frequencies above 0 Hz to highly correlate with the steady-state IV curve. Likewise, measurements in the presence of the noise function demonstrate that the noise function does not cause any frequencies above 0 Hz to correlate with the steady-state IV curve when no ion channels are present. Based on this verification, the method can now be applied to preparations containing a single ion channel type with the intent of identifying frequencies whose amplitudes correlate specifically with that channel type.

Watch this Scientific Journal Video about Recapitulation of an Ion Channel IV Curve Using Frequency Components at JoVE.com

Recapitulation of an Ion Channel IV Curve Using Frequency Components

There are technical obstacles to measuring current flux through multiple ion channels simultaneously, and later discerning what portion of the transmembrane current is due to each channel type. To address this need, this method presents a way to generate the IV curve of individual channel types using specific frequency components.

INTRODUCTION: Presently, there are no established methods to measure multiple ion channel types simultaneously and decompose the measured current into ...

recapitulation-of-an-ion-channel-iv-curve-using-frequency-components

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13.5K Views.  University of Utah. There are technical obstacles to measuring current flux through multiple ion channels simultaneously, and later discerning what portion of the transmembrane current is due to each channel type. To address this need, this method presents a way to generate the IV curve of individual channel types using specific frequency components.

Video: Recapitulation of an Ion Channel IV Curve Using Frequency Components

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

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.

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

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

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.

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