This work was supported by National Science Foundation grant MCB-0951199, and in part by the NIH grant EY01910, California Institute of Regenerative Medicine grants RB1-01417 and by the Funda&#231;&#227;o para a Ci&#234;ncia e Tecnologia (FCT) grant SFRH/BD/87256/2012.

1. Ion-selective Self-referencing Microelectrode Preparation
<ol>
	<li>Preparation of ion-selective microelectrode
	<ol>
		<li>Heat pull thin walled borosilicate capillaries without filament (1.5 mm outer diameter, 1.12 mm inner diameter) using a microelectrode puller. 
		Note: This gives tips 3-4 &#181;m in diameter. Smaller tips have higher resistance which makes microelectrodes more susceptible to electronic noise and is also associated with a slower response to a change in ion concentration. Useful information can be found in the paper published by Smith et al.26.</li>
		<li>Silanize the electrodes to render the inner surface hydrophobic to aid retention of the lipophilic ionophore cocktail. Place the microelectrodes in a metal rack and heat O/N in an oven at &#62;100 &#176;C to dry them. The rack is a metal plate with 2 mm diameter holes drilled part of the way through. Place the electrodes in the holes tip upwards with a 250 ml glass beaker over them.</li>
		<li>In the morning, turn the oven off and while wearing insulated gloves, carefully remove the metal rack with the electrodes and beaker in place. Close the oven door to retain the heat.</li>
		<li>Wear latex or nitrile gloves, lab coat and eye protection. With a plastic Pasteur pipette, place a drop of silanization solution I at the base of each electrode (keep the beaker in place; use the pouring lip for pipette access). The silanization solution is vaporized by the hot plate and silanizes the inside of the electrodes. Use a chemical extractor fume hood for this stage. Place the rack/beaker/electrodes back in the hot oven for a few hours to allow any remaining silanization solution to evaporate. 
		Note: For safety reasons, do not turn the oven back on. Place a label on the oven indicating it must not be switched on as it may contain harmful and flammable vapor.</li>
		<li>After cooling, store the microelectrodes in a microelectrode storage jar inside a glass desiccator with 400 g of desiccant. Microelectrodes can be stored thus for many weeks. 
		Note: An alternative silanization method is described in Smith et al.26</li>
		<li>Back-fill the microelectrode with 50 to 100 &#181;l (a length of about 1 cm) of a solution containing 100 mM of the ion to be measured (see Table 1 and Figure 1B). Use a disposable plastic Pasteur pipette heat pulled in a Bunsen burner to a fine filament. Rinse the pipette in dH2O afterward to prevent blockage. 
		Note: Alternatively, adjust the ion concentration of the backfilling solution to match the concentration of ion in the external solution27.</li>
		<li>Observe the microelectrode under a dissecting microscope to ensure the absence of air bubbles.
		<ol>
			<li>If bubbles are present tap the microelectrode lightly with a finger nail while holding the electrode vertically (tip down) and/or push the bubbles out the tip by applying back pressure using a syringe modified with a silicone tube replacing the needle.</li>
		</ol>
		</li>
		<li>Tip-fill the microelectrode with 15 to 20 nl (a length of 30-50 &#181;m) of ion-specific ionophore cocktail (see Table 1). Place a small droplet of ionophore cocktail on the short edge of a microscope slide. Observe the microelectrode tip under a dissecting microscope and move it toward the microscope slide until the microelectrode tip touches the ionophore cocktail for only about half a sec. Draw the ionophore cocktail into the microelectrode by capillary pressure. 
		Note: Avoid a long column of ionophore cocktail as this increases the probe&#8217;s electrical resistance which can make it susceptible to electronic interference (noise) and also slows the response time.</li>
		<li>Mount the microelectrode in a straight microelectrode holder with a gold 1 mm male connector and AgCl(Ag+) wire (Figure 1B).</li>
		<li>Attach the microelectrode holder to the head stage mounted on a three-dimensional computer-controlled electronic micropositioner (Figure 1A).</li>
		<li>Place the microelectrode tip in measuring solution appropriate for the sample to be measured (physiological saline, culture medium, etc.) to allow the microelectrode to stabilize for an hr or two, or even overnight.</li>
	</ol>
	</li>
	<li>Preparation of the reference electrode
	<ol>
		<li>Reference electrodes (Figure 1C) are the same capillaries as above. Cut the capillary with a diamond pencil into 5 cm lengths and fire-polished at each end for 1-2 sec&#160;in a Bunsen flame.</li>
		<li>Fill these electrodes with ~200 &#181;l of a 3 M solution of NaCl, CH3CO2K (potassium acetate) or KCl with 2% agarose. Choose the solution depending on the ion to be measured (the reference electrode should not contain the ion being measured; see Table 1). Mix the agarose and the solution and heat to almost boiling in a microwave. Stir to dissolve the agarose (the solution goes clear).</li>
		<li>Attach the reference electrode to a plastic Pasteur pipette and draw the hot solution into the capillary.</li>
		<li>Drop the electrode into cold 3 M NaCl, CH3CO2K or KCl solution and store in this 3 M solution in sealed tubes prior to use. Discard any reference electrodes with air bubbles.</li>
		<li>Mount the reference electrode in a straight microelectrode holder (pre-filled with 3 M solution) with an AgCl(Ag+) pellet inside and a gold 2 mm male connector (Figure 1C) and attach the electrode and holder onto a manual micro-positioner mounted on a magnetic stand.</li>
	</ol>
	</li>
</ol>
2. Ion-selective Self-referencing Microelectrode Calibration
<ol>
	<li>Preparing calibrating solutions containing the ion of interest as in the reference solution; see Table 1. Bracket the concentration that is in the solution the sample will be in (e.g. culture media, physiological saline). That is, one calibration solution must contain a lower concentration of ion than in the measuring solution, and one higher.
	<ol>
		<li>For example, use saline that contains 1 mM of K+. To bracket this concentration, dissolve KCl powder in deionized water to a concentration of 10, 1 and 0.1 mM in serial dilutions. Use these calibration solutions. Alternatively, use at least two of these solutions.</li>
	</ol>
	</li>
	<li>Immerse the ion-selective microelectrode and the reference electrode in each calibration solution and let the voltage value stabilize for 1 to 3 min&#160;before recording the corresponding voltage using the dedicated software (see Table 1).</li>
	<li>As the software saves the data (amplifier output) as a txt file, copy the data into a spreadsheet file. Plot the microelectrode output (mV) against the logarithm of the molar ion concentration (Figure 2A).</li>
	<li>Apply a linear regression and calculate the Nernst slope, intercept and R2 value. Accept the microelectrode if the Nernst slope is 58 &#177; 11 mV/decade for monovalent ions and 29 &#177; 11 mV/decade for divalent ions (for cations, the Nernst slope is positive, for anions it is negative). Additionally, good microelectrodes should have a strong linear correlation (R2 &#62;0.9; Figure 2B). 
	Note: The mV output of the amplifier used here gives mV reading with a tenfold gain. Values have to be divided by a factor of ten.</li>
	<li>Use the linear regression formula to convert the raw mV output of the microelectrode into actual ion concentration (Figure 2B).</li>
</ol>
3. Validation of the Ion-selective Microelectrode Technique
<ol>
	<li>Preparing an artificial source
	<ol>
		<li>Artificial source capillaries are the same capillaries as above. Heat pull the capillary using a microelectrode puller as in step 1.1.1.</li>
		<li>Backfill these capillaries with 200 &#181;l of a 1 M solution of NaCl, KCl, CaCl22 H2O or pH 4 buffer. Choose the artificial source solution depending on the ion to be measured (see Table 1). 
		Note: Alternatively, pull electrodes with bigger tip diameter (~20 &#181;m) and tip-fill with the same solutions but containing 0.5-1% agarose (agarose will prevent any bulk flow of solution).</li>
		<li>Mount the artificial source capillary on a micromanipulator and immerse it in the solution used to measure the flux of the ion in the samples. Leave the artificial source in the solution for 30 min&#160;to 1 hr to allow stabilization of the gradient.</li>
	</ol>
	</li>
	<li>Validation of the ion-selective microelectrode
	<ol>
		<li>Immerse the ion-selective microelectrode about one centimeter away from the artificial source capillary in the solution used to measure the flux of ion on the samples and close the circuit with the reference electrode as before. Let the voltage value stabilize for 1 to 3 min&#160;before recording the corresponding voltage using the dedicated software for 1 to 2 min. This value corresponds to the buffer value (in literature also referred as reference, background or blank value).</li>
		<li>Move the ion-selective microelectrode to about 5 &#181;m from the artificial source and let the voltage value stabilize for 1 to 3 min&#160;before recording the corresponding voltage using the software for 1 to 2 min.</li>
		<li>Repeat the above procedure by placing the ion selective microelectrode at 10, 20, 40, 80, 160, 320, 640 and 1280 &#181;m away from the artificial source capillary.</li>
		<li>Extract the data as a txt file and copy the values into a spreadsheet file.</li>
	</ol>
	</li>
	<li>Calculate the ion concentration corresponding to the mV values in the same way as for the calibration values. Plot the value. 
	Note: If an ion flux is present, the microelectrode detects a difference in ion concentration between the two positions (Figure 3B). If the artificial source contains more ions of the species measured than the solution, the concentration should be higher close to the source than far away, validating the ability of the ion-selective microelectrode to correctly detect the direction of an ion flux (in this case efflux; for an artificial sink, with lower specific ion concentration than measuring medium, it should be influx).
	<ol>
		<li>Calculate the ion flux using Fick&#8217;s law of diffusion: J = c &#181; (dc/dx) where c is the ion concentration in the solution (mol cm-3), &#181; is the ion mobility (mol cm N-1 sec-1), and dc is the concentration difference over distance dx (cm) (Figure 2C). Ion flux data are usually presented in pmol cm-2 s-1or nmol cm-2 sec-1. 
		Note: An alternative method of ion flux calculation described by Smith et al.26. can be used. Main differences include the use of the diffusion coefficient instead of the ion mobility and the subtraction of the background ion flux (also voltage drift or correction factor) calculated from measurement of ion flux in saline solution without sample.</li>
		<li>Plot the mean of the ion fluxes of each step against the distance from the source (Figure 2D). Moving away from the source, observe an exponential decrease of the flux value validating the ability of the ion-selective microelectrode to sense different magnitude of ion fluxes.</li>
		<li>Do the artificial source validation once for each specific ion intended to be recorded in order to validate its correct direction and magnitude measurements with a large signal-to-noise ratio. 
		Note: Ion flux measurement of the buffer without samples indicates the background level or noise. Typically, buffer measurement shows no clear fluctuation of the ion concentration leading to very small flux that displays variable directions.</li>
	</ol>
	</li>
</ol>
4. Preparation of Measuring Chamber
Note: Before experiments, consider the sample to be measured and how the sample is to be mounted and immobilized for microelectrode measurements.
<ol>
	<li>For Xenopus laevis oocyte measurements cut a 1 cm square of an 800 &#181;m nylon mesh (nitex mesh) and glue it into a plastic Petri dish (Figure 1E).</li>
</ol>
5. Ion Flux Measurement
<ol>
	<li>Measure the ion concentration present in the buffer used to perform the measurements on the sample in the same way as for the calibration solution. X. laevis oocytes require Mark&#8217;s Modified Ringer (MMR). Dissolve NaCl, KCl, CaCl, MgCl and HEPES into deionized water to reach a final concentration of (mM): 100 NaCl, 2 KCl, 2 CaCl, 1 MgCl and 5 HEPES. Adjust the pH of the buffer to 7.5 using NaOH.</li>
	<li>Place the sample into the measuring chamber and bring the ion-selective microelectrode close to the sample (about 10 &#181;m away) using the micropositioner to define the close position of the microelectrode (Figure 3A).</li>
	<li>Start the low frequency (0.3 Hz) excursion (100 &#181;m) of the microelectrode between the close position and a position away from the sample (distant) using the dedicated software. Ensure that the movement of the microelectrode is perpendicular to the surface of the sample. 
	Note: The excursion of the microelectrode can be set on the software. Large excursion increases the gradient read allowing an easier detection of small fluxes during the measurement while lengthens the sampling interval and decreases the temporal resolution. See Figure 3A for an example.</li>
	<li>Start the recording using the software. The microelectrode pauses at each position and the electric potential in mV is recorded on the computer. Obtain measurements for at least 2 min, allowing signal stabilization. For short time-lapse experiments, record potential variations at the position of interest for the whole time course.</li>
	<li>Extract the data as a txt file and copy the values into a spreadsheet file.</li>
	<li>Calculate the ion concentration corresponding to the mV values in the same way as for the calibration values. Plot the value. 
	Note: If an ion flux is present, the microelectrode detects a difference in ion concentration between the two positions (Figure 3B).</li>
	<li>Calculate the ion flux using Fick&#8217;s law of diffusion as before (step 3.3.1).</li>
	<li>Repeat the buffer measurement before measuring a new sample and repeat the procedure of flux measurement and calculation for every new sample.</li>
</ol>
6. Statistical Analysis and Data Presentation
<ol>
	<li>Test the independent effects of the position and/or of the time on ion fluxes under the control condition using an ANCOVA model with mixed effects28. 
	Note: Analysis of covariance (ANCOVA) is a general linear model that mixes regular ANOVA and regression by allowing both categorical and continuous measures to be used as independent variables. In addition, in the presence of correlated errors induced by repeated measures per individual and eventual nested effects, mixed effects models are used to model accurate estimates of both fixed and random effects.</li>
	<li>Calculate pairwise comparisons using Student t-test between group levels with Bonferroni correction for multiple testing28.</li>
	<li>Generate boxplots to summarize ion flux measurements according to position and time. Include p values from the pairwise Student t described above (Figure 3D) and indicate significance levels of p values as follows: *: p &#60;0.05; **: p &#60;0.01; ***: p &#60;0.00129</li>
</ol>

The authors declare that they have no competing financial interests.

The most critical steps for successful measurement of extracellular ion fluxes in vivo are: the reduction of the noise, the correct fabrication of the ion-selective microelectrodes and reference electrode, and the positioning of the sample and both electrodes.
In order to minimize the noise, the recording system should be in an earthed (grounded) Faraday cage preferably with a metal-topped (vibration isolation) table which is also earthed. In addition, the microscope chassis should al...

All animal cells are surrounded by a lipid bilayer membrane which separates the cytoplasm from the outside environment. The cell maintains an electrical membrane potential, negative inside, by active transport of ions1. The membrane potential is a stored energy source which the cell can utilize to operate various molecular devices in the membrane2. Neurons and other excitable cells have large membrane potentials. Rapid opening of sodium channels collapses the membrane potential (depolarization) and produces the action potential which is transported along the length of the neuron2. Aside from these rapid electrical changes, many tissues and organs generate and maintain significant long-term electrical potentials. For example, skin and corneal epithelia generate and maintain trans-epithelial potentials and extracellular electric currents by directional pumping of ions (mainly sodium and chloride)3.
While measurements of endogenous extracellular electric current using the vibrating probe4-6 and measurements of membrane or trans-epithelial potentials using the microelectrode system7-10 allow measurement of the electric parameters of cell membranes and epithelial cell layers, they give no indication of the ion species involved.
Microelectrodes with selective ionophore can measure specific ion concentration in solution. Ion gradients or flux could be measured with two or more electrodes at different positions. However, the intrinsic voltage drift of each probe would be different, causing inaccurate measurements or even detection of a gradient that was not present. A single electrode used in &#8220;self-referencing&#8221; mode whereby it moves at low frequency between two points solves this problem. Now the ion flux can be seen against the background of a relatively slow and stable signal drift (see Figure 3B).
The ion-sensitive measuring system uses ion-selective self-referencing microelectrodes to detect small extracellular fluxes of ions close to tissues or single cells. The system consists of an amplifier which processes the signal from the microelectrode and a micro stepper motor and driver to control the motion of the microelectrode. The ion-selective microelectrode and the reference electrode that close the circuit are connected to the amplifier via a headstage pre-amplifier (Figure 1A). Computer software determines the parameters of the microelectrode movement (frequency, distance) and also records the output of the amplifier. The stepper motor controls the microelectrode movement via a three-dimensional micropositioner. A low frequency vibrating ion-selective microelectrode was first developed in 1990 to measure specific calcium flux11. As well as calcium, commercially accessible ionophore cocktails are now available to make microelectrodes sensitive to sodium, chloride, potassium, hydrogen, magnesium, nitrate, ammonium, fluoride, lithium or mercury.
Basically, the self-referencing ion-selective microelectrode technique converts the activity of a specific ion dissolved in a solution into an electric potential, which can be measured by a voltmeter. The ionophore cocktail is an immiscible liquid (organic, lipophilic) phase with ion-exchange properties. The ionophore selectively complexes (binds) specific ions reversibly and transfers them between the aqueous solution contained in the microelectrode (electrolyte) and the aqueous solution in which the microelectrode is immersed (Figure 1D). This ion transfer leads to an electrochemical equilibrium and a variation of the electric potential between the microelectrode and the reference electrode is measured by the voltmeter. The voltage is proportional to the logarithm of the specific ion activity according to the Nernst equation allowing the calculation of the ion concentration (Figure 2A and B).
At present, several systems allow measurement of ion flux using a similar concept or principle. For example, the Scanning Ion-selective Electrode Technique (SIET)12,13 or the Microelectrode Ion Flux Estimation (MIFE) technique developed by Newman and Shabala14-16 are commercially available and widely used by the research community in order to determine specific ion fluxes occurring at cell membrane and tissue across a variety of animal, plant and single living cell models. Ion-selective microelectrodes have been used to measure hydrogen, potassium and calcium flux across plant roots17, chloride flux in rat cerebral arteries18 and in pollen tubes19, hydrogen flux in skate retinal cells20, calcium flux in mouse bone21, various ion fluxes in fungal hyphae22 and in rat cornea23, and finally calcium flux during single cell wound healing12,24. See also the following review for detailed information on ion-selective self-referencing microelectrodes25.
The following article describes in detail how to prepare and perform measurement of endogenous extracellular ion fluxes using the ion-selective self-referencing microelectrode technique at the single cell level.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>IonAmp&#160;</td>
			<td>&#160;BioCurrents Research Center, Woods Hole, MA, USA</td>
			<td>none</td>
			<td>amplifier created by the BioCurrents Research Center, Woods Hole, MA, USA; Similar system can be purchased from &#8220;XBL function matters&#8221; (http://www.xuyue.org/) or from &#8220;YoungerUSA&#8221; (http://www.youngerusa.com/) or from Applicable Electronics(http://www.applicableelectronics.com/)</td>
		</tr>
		<tr>
			<td>IonAmp32&#160;</td>
			<td>&#160;BioCurrents Research Center, Woods Hole, MA, USA</td>
			<td>none</td>
			<td>software created by the BioCurrents Research Center, Woods Hole, MA, USA; Similar system can be purchased from &#8220;XBL function matters&#8221; (http://www.xuyue.org/) or from &#8220;YoungerUSA&#8221; (http://www.youngerusa.com/) or from Applicable Electronics(http://www.applicableelectronics.com/)</td>
		</tr>
		<tr>
			<td>Headstage pre-amplifier</td>
			<td>&#160;BioCurrents Research Center, Woods Hole, MA, USA</td>
			<td>INA116</td>
			<td>BSR Voltage Follower INA116, designed by the BioCurrents Research Center, Woods Hole, MA, USA; Similar system can be purchased from &#8220;XBL function matters&#8221; (http://www.xuyue.org/) or from &#8220;YoungerUSA&#8221; (http://www.youngerusa.com/) or from Applicable Electronics(http://www.applicableelectronics.com/)</td>
		</tr>
		<tr>
			<td>MicroStep Driver</td>
			<td>&#160;BioCurrents Research Center, Woods Hole, MA, USA</td>
			<td>none</td>
			<td>three MicroStep drivers are required for X, Y and Z-positioning; created by the BioCurrents Research Center, Woods Hole, MA, USA; Similar system can be purchased from &#8220;XBL function matters&#8221; (http://www.xuyue.org/) or from &#8220;YoungerUSA&#8221; (http://www.youngerusa.com/) or from Applicable Electronics(http://www.applicableelectronics.com/)</td>
		</tr>
		<tr>
			<td>Manual micropositioner&#160;&#160;</td>
			<td>World Precision Instruments&#160;</td>
			<td>Model KITE-R</td>
			<td>Similar system can be purchased from Applicable Electronics(http://www.applicableelectronics.com/)</td>
		</tr>
		<tr>
			<td>Magnetic stand&#160;&#160;&#160;</td>
			<td>World Precision Instruments</td>
			<td>Model M10</td>
			<td>Similar system can be purchased from Applicable Electronics(http://www.applicableelectronics.com/)</td>
		</tr>
		<tr>
			<td>Vibration isolation table&#160;&#160;</td>
			<td>Newport Inc.&#160;&#160;&#160;&#160;&#160;</td>
			<td>Model VW-3036-OPT-023040</td>
			<td>Similar system can be purchased from Applicable Electronics(http://www.applicableelectronics.com/)</td>
		</tr>
		<tr>
			<td>Part of three dimentional micropositioner: angle bracket, 90&#176;, slotted faces</td>
			<td>Newport Inc.&#160;&#160;&#160;&#160;&#160;</td>
			<td>Model 360-90</td>
			<td>Assemblage of the three dimantionnal micropositionner requires also Three electric rotary motors for X, Y, Z control, MPH-1 mounting arm with MCA-2 adjustable-angle post and Various Newport connectors and screws to bolt onto vibration table</td>
		</tr>
		<tr>
			<td>Part of three dimentional micropositioner: Peg-Joining Dovetail Stage 0.5 inch X Travel</td>
			<td>Newport Inc.&#160;&#160;&#160;&#160;&#160;</td>
			<td>460PD-X</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Part of three dimentional micropositioner: Quick-Mount Linear Stage, 0.5 inch XY Travel</td>
			<td>Newport Inc.</td>
			<td>460A-XY</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Kwik-Fil thin walled borosilicate glass capillaries without filament&#160;</td>
			<td>World Precision Instruments&#160;</td>
			<td>TW150-4</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Electrode puller&#160;</td>
			<td>Narishige</td>
			<td>&#160;PC-10</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Metal rack</td>
			<td>Made in-house</td>
			<td>none</td>
			<td>Metal electrode holder made in-house by drilling 2 mm wide holes half centimeter spaced in a 10cm by 15cm rectangular base of steel</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>QL</td>
			<td>Model 10 Lab Oven</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Silanization solution I&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>85126</td>
			<td>Hazardous, handle as recommended by provider&#160;</td>
		</tr>
		<tr>
			<td>Glass Petri dish; Pyrex</td>
			<td>Fisher Scientific</td>
			<td>316060</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Electrode/micropipette storage jar</td>
			<td>World Precision Instruments&#160;</td>
			<td>E215</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Glass dessicator</td>
			<td>Fisher Scientific</td>
			<td>08-595E</td>
			<td>Contains Drierite dessicant (W.A. Hammond Drierite Co. Ltd, Xenia, OH, USA). Place petroleum jelly on the seal to make it airtight.</td>
		</tr>
		<tr>
			<td>Plastic Pasteur pipette&#160;</td>
			<td>Fisher Scientific</td>
			<td>11597722</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td>Fisher Scientific</td>
			<td>S97329</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Sigma-Aldrich</td>
			<td>S8902</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Straight microelectrode holder</td>
			<td>Warner Instruments</td>
			<td>QSW-A15P</td>
			<td>with a gold 1 mm male connector and Ag/AgCl wire</td>
		</tr>
		<tr>
			<td>Straight microelectrode holder&#160;</td>
			<td>World Precision Instruments</td>
			<td>MEH3S</td>
			<td>with a AgCl(Ag+)pellet inside and a gold 2 mm male connector&#160;</td>
		</tr>
		<tr>
			<td>6 cm Petri dish</td>
			<td>VWR</td>
			<td>60872-306</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Nitex mesh</td>
			<td>Dynamic Aqua-Supply Ltd.</td>
			<td>NTX750</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Glue; Loctite epoxy</td>
			<td>VWR</td>
			<td>500043-451</td>
			<td>Mix glue and hardener in equal parts in a plastic weighing boat and mix thoroughly. Sets quickly but leave at RT for 24 h for full curing</td>
		</tr>
		<tr>
			<td>Deionized water&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>99053</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Sodium Hydroxyde</td>
			<td>Sigma-Aldrich</td>
			<td>S8045</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Potassium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>P1190</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td>none</td>
		</tr>
	</tbody>
</table>

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.

We have previously shown that calcium influx appears after single cell wounding24. We therefore asked whether other ion fluxes occur upon single cell wounding. We used the X. laevis oocyte, a well-established model for single cell wound healing30-34 and electrophysiological recording24,35-39. Interestingly, potassium ions are more concentrated inside X. laevis oocytes (about 110 mM)40 than in the extracellular solution used (in MMR 1x: 1 mM) suggesting an effl...

Watch this Scientific Journal Video about Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique at JoVE.com

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

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 ...

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Biology

13.9K Görüntüleme.  University of California, Davis. 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.

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

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 ...

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.

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 ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

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 ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...