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

The overall goal of this procedure is to measure extracellular ion fluxes in vivo using the ion selective self-referencing micro electrode technique. This is accomplished by first assembling the ion selective and the reference micro electrodes. The second step is to calibrate and validate the ion selective self-referencing micro electrodes.

Next, the ion specific flux measurements are made on the biological sample in the form of voltage recordings. Finally, the voltage recording is converted into ion flux. Ultimately, this technique allows accurate non-invasive measurement of specific extracellular ion fluxes in living cells or tissues.

These method can help answer key questions in physiology or aing as well as regeneration or pathology. For example, this method allows the identification of specific ion fluxes, which control biological processes such as tissue morphogenesis growth or differentiation. Demonstrating this procedure will be Dr.Gim Ardi, a postdoc, and Dr.Benjas laboratory Prior to electrode calibration mount, a pre-filled ion selective electrode in a micro electrode holder equipped with a one millimeter gold male connector and a silver chloride coated silver wire.

Then attach the micro electrode holder to a three axis computer controlled electronic micro positioner. The ion selective electrode itself should already contain the electrolyte and the ion four cocktail solution pertaining to the ions of interest. Once mounted, the electrode should be immersed in a solution containing one millimolar potassium chloride.

In addition, mount the reference electrode in a straight micro electrode holder containing a three molar salt solution In agar, a silver chloride coated silver pellet and a two millimeter gold male connector for the reference electrode. The choice of the salt solution depends on the ion species to be measured. After preparing the reference electrode, attach the electrode holder to a manual micro positioner that is prem mounted onto a magnetic stand.

Once mounted, the electrode should be immersed in a solution containing one millimolar potassium chloride. Before starting, the ion selective electrode setup have prepared a dilution series of electrolyte calibration standards containing the ions of interest. For instance, if potassium cation are to be detected at a physiological concentration of around one millimolar, make a three log series of potassium chloride solutions at ten one and 0.1 millimolar.

That maximizes the dynamic range of detection that the assay requires. To calibrate the ion selective electrode, start by immersing both the ion selective and reference electrodes into the least concentrated calibrating solution in this case, 0.1 millimolar. After a brief stabilization period of one to three minutes at room temperature, measure and record the steady state voltage.

Repeat the voltage measurements for the rest of the calibration standards, plop the measured voltages against the logarithm of the molar ion concentrations. For each standard solution, apply linear regression to ascertain the N slope intercept and are squared values of the calibration curve. To calculate the nert slope, remember to divide the measured voltages by the total gain of the voltage amplifier.

To demonstrate the measurement protocol, the flx of potassium ions from a perforated frog oocyte will be characterized using a potassium sensitive micro electrode Before starting the ion selective electrode setup, cuss a one centimeter patch from an 800 micron nylon mesh and glue it to a plastic Petri dish. Also, have prepared beforehand mark's modified ringer solution with the following formulation, 100 millimolar sodium chloride, two molar potassium chloride, two millimolar calcium chloride, one millimolar magnesium chloride, and five millimolar of he peas. Adjust the pH of the buffer to 7.5 with sodium hydroxide and add an aliquot of the completed buffer into the Petri dish.

Carefully place the frog cyte the ion selective electrode and the reference electrode into the Petri dish. Then perforate the cyte and actuate the micro positioner, such that the ion selective electrode is about 10 microns away from the sample. Nomenclature wise, the current distance between the sample and the electrode will be designated as the close position.

Next, actuate the micro positioner such that the ion selective electrode periodically transverses from the close position of 10 microns to a more distant position of 100 microns, and then returns to the close position at a rate of 0.3 hertz, or in other words, three complete journeys for every 10 seconds. During micro positioner, actuation verify that the movement of the electrode is truly perpendicular to the wounded tangential surface of the cyte activate the voltage measurement and recording software. The micro positioner is programmed to pause briefly at the close and distant positions while voltage data are being collected.

Data is recorded continuously during oocyte wound healing, which takes around 15 minutes for longer experiments, such as observing multi-day process during opus tadpole tail regeneration measurements are recorded for two minutes at different time points. For example, 1, 3, 6, 12, and 24 hours. Upon completion of the experiment, extract the voltage data as a text file and copy the values into a spreadsheet.

Use the previously determined calibration curve to convert the measured voltage values into their corresponding ion concentrations. Plot the extrapolated ion concentrations as a function of distance away from the biological sample. Finally, calculate the ion flux using fixed law of diffusion.

Where C is the ion concentration in the solution, MU is the ion mobility, and DC DX is the concentration difference over distance dx. Prior to biological experimentation, the value of the NER slope intercept and the entire calibration curve for the ion selective electrode can be validated against an artificial ion gradient generated from a one molar ion source placed in a Petri dish. When changes in potassium ion concentrations are plotted at a distance close to or distant from annu site wound.

The data suggests that a massive potassium flx event occurs at onset of cellular wounding as wound healing progresses, the potassium flx gradually abates back to quiescent levels after 15 minutes when the wound is fully healed. Furthermore, when the measured potassium ion concentrations are converted to ion flux, the sudden increase and the gradual decrease of potassium flx after wounding appears to be a statistically significant effect when compared to an intact control oocyte. Following this procedure, other methods such as fluorescent bioelectricity labeling can be performed in order to answer additional questions on the characteristics of intercellular and subcellular ion fluxes.