Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

The overall goal of this procedure is to measure trans epithelial fluid secretion and ion flux rates in the drosophila melan gaster renal tubule, also known as the akian tubule. This method can help answer key questions in the field of epithelial ion transport, such as the molecular characterization of basic transport mechanisms and their regulation. The main advantage of this technique is that it combines the powerful genetics of drosophila melanogaster with the accessibility of its renal tubules to physiological study.

This method can also provide insight into epithelial ion transport mechanisms and other insects such as the disease vectors. Rodneys Xis IDs I gti and anno Gambier. First prepare a reusable glass rod to transfer the tubules using a glass cutter and wearing safety equipment.

Cut a three millimeter thick black stained glass into strips that are six millimeters wide and 10 centimeters long using bun and burner flame. Soften the short end of two strips, then press the soft ends together and swiftly pull them apart to produce a fine glass rod with a handle. To prepare a potassium ion specific electrode or ISE load a one milliliter syringe with 0.5 molar potassium chloride and backfill o acidized pipette.

Make sure no air bubbles are trapped, which may require microscopic examination. Use a gentle flick to the pipette to remove any trapped air. Next, under a hood, attach the ISE to a suction device and secure the ISE on molding clay on an inverted 3.5 centimeter Petri dish.

Then point the off of the stopcock handle to the side port and draw back on the syringe to generate negative pressure on the tip of the ISC. When completed, turn the handle so the off is pointing to the tubing. Next, using safety precautions, dip the small opening of a 10 microliter pipette tip into ion four solution.

Then by placing a gloved finger over the larger opening, expel a small drop of ion four solution onto the outside surface of the pipette. Now very carefully touch the drop of solution to the tip of the ISC. Do not touch the tip of the ISE with a pipette tip to avoid breaking the ISE tip.

When completed, turn the handle so the off is pointing towards the side port next under a microscope at 40 x magnification. Check if the solution was taken up into the ISE. If the ISE is optimally siliconized, the interface between the IOR and the backfill solution should be flat.

Now partially fill a beaker with 150 millimolar potassium chloride. Then place the ISE tip down onto the wall of the beaker, which has molding clay to secure the ISE in place. Next, prepare the reference electrode.

Use a micro filament and syringe to fill the tip and shank of the reference electrode with one molar sodium acetate. Gently flick the pipet to remove any air bubbles with the same technique. Use a second micro filament and syringe to backfill the same electrode with three molar potassium chloride.

Store the reference electrode alongside the ISE after preparing the electrodes and at least 30 minutes before dissection. Ready some bathing medium and oph as saline to allow them to warm to room temperature prior to dissections. Next, calibrate the ISC to see if it is working onto a silicone elastomer coated Petri dish.

Make two replicate rows of potassium chloride drops. Drop four different concentrations in 0.6 microliter drops. Then carefully layer two milliliters of mineral oil over the drops.

Now in a faraday cage, place the dish under a dissecting scope. Next, thread the reference electrode and ISC over the silver wires and fasten them onto micro electrode holders with the electrometer in standby mode, use the micro manipulators to advance the ISC and reference electrode onto either side of the 15 millimolar potassium chloride drop to be measured. Then advance into the drop.

Switch the electrometer to operate and let the reading stabilize. Then record the reading. Switch the electrometer back to stand by.

Withdraw the electrodes from the 15 millimolar potassium chloride drop. Move the dish so that the 75 millimolar potassium chloride drop is in the center of the field and advance the electrodes into the 75 millimolar potassium chloride drop. Switch the electrometer to operate.

Let the reading stabilize and record the potential for the 75 millimolar potassium chloride drop. Switch the electrometer back to stand by. Then withdraw the electrodes from the 75 millimolar potassium chloride drop.

Move the dish so that the 150 millimolar potassium chloride drop is in the center of the field and advance the electrodes into the 150 millimolar potassium chloride drop. Switch the electrometer to operate. Let the reading stabilize and record the potential for the 150 millimolar potassium chloride drop.

Then withdraw the electrodes from the 150 millimolar potassium chloride drop. Move the dish so that the 200 millimolar potassium chloride drop is now in the center of the field and advance the electrodes into the 200 millimolar potassium chloride Drop, measure and record the potential for the 200 millimolar potassium chloride drop. Then return the electrometer to stand by immediately prior to the dissections.

View the assay dish at 10 x magnification and add enough SBM to just fill each well of the assay dish. Avoid overfilling. Next carefully.

Layer about 12 or 13 milliliters of mineral oil over the wells to completely cover the SBM. Use a 20 gauge needle to pop the air bubbles. Now aesthe ties the flies with carbon dioxide and place one backside down onto a silicone elastomer coated dish.

Secure the fly with a minuchin pin and immerse the fly in room temperature Rees sous saline. Use forceps in the nondominant hand to hold the abdomen at the thoracoabdominal junction. Then with another pair of forceps in the dominant hand, peel the abdominal cuticle away.

Start at the theso abdominal junction and move towards the tail end of the fly. The gut with attached male piggy and tubules will be exposed with this maneuver without touching the tubules. Dissect free the gut and attach tubules.

Secure the gut with the non-dominant hand and use a 30 gauge needle to sever the ureter from the gut with the dominant hand. It is essential that no tears or rinse be introduced into the tubule other than at the ureter. Next, using a fine glass rod, transfer a tubule pair to a well of the assay dish immediately after the transfer.

Pick up the end of one of the tubules with the rod. Withdraw it from the bath until the cut end of the ureter is halfway between the pin and the bathing drop. Then wrap the end of the tubule around.

A minuchin pin. Fluid will subsequently be secreted from the cut end of the ureter. Write down the well tubule information such as genotype or condition and the time.

This is the start time of secretion. Then proceed with the next dissection. At the desired time point, prepare to measure the first secreted fluid drop.

For example, if you are interested in measuring a two hour time point, this would be 120 minutes after the start time for the first drop under sufficient magnification. First, measure the diameter of the drop with an ocular micrometer. Note the magnification used the diameter and the time.

Second, measure the ion activity. Advance the ISE and reference electrode into the fluid drop. Then switch the electrometer to operate.

Allow the reading to stabilize and record the value. Then switch the electrometer back to standby. Withdraw the electrodes from the drop.

Continue with the next drop at the appropriate time. For example, 120 minutes after the start time. For the second drop, measure the diameter using the ocular micrometer and measure the potential with the electrodes.

With the electrometer in operate mode. Then switch the electrometer back to standby and withdraw the electrodes from the secreted fluid drop. Proceed to the next drop at the appropriate time.

The Ramsay assay with ion specific electrodes was used to measure sodium and potassium ion fluxes information that is not captured by measuring fluid secretion rates alone decreased fluid secretion in tubules from flies. Carrying a homozygous null mutation in NKCC was found to be driven by a decrease in potassium flux. In those same flies, there was no change in sodium flux application of the sodium potassium APAs inhibitor.

WBA had distinct effects on each of the three measured parameters. In wild type two ues, the fluid accretion rate was unchanged, but sodium flux increased. At the same time potassium flux decreased.

Furthermore, w Bain had no effect on trans epithelial potassium flux in the NKCC null flies Once mastered 15 to 20 tubules can be analyzed in a single day's experiment, which takes about five to six hours from beginning to end. It's important to remember to carefully wash the assay dish at the end of the day to make sure residual salts in the wells do not alter the ionic and osmotic composition of the bathing saline in subsequent experiments. Once the experimenter is comfortable with the technique, permutations can be introduced to probe the underlying biology of epithelial ion transport, such as analyzing tubules of differing genotypes to determine the role of a specific transporter or signaling pathway.

After watching this video, you should have a good understanding of how to measure trans epithelial fluid secretion and ion fluxes in dissected drosophila renal tubules. Don't forget that working with sharps, nitric acid di chloral dimethyl saline and potassium ionophore can be extremely hazardous precautions, such as using appropriate personal protective equipment working in the hood when appropriate, and following manufacturers and institutional safety guidelines can maximize safety.