Name: Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli (Video) | JoVE Journal
Uploaded: 2015-06-27T14:00:00.000+00:00
Duration: 12 min 19 s
Description: Learn calcium imaging and single-channel analysis in podocytes using freshly isolated rat glomeruli. This method enables real-time monitoring of intracellular calcium dynamics and ion channel activity for kidney research.

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5 minutes

The authors would like to thank Glen Slocum (Medical College of Wisconsin) and Colleen A. Lavin (Nikon Instruments, Inc.) for excellent technical assistance with microscopy experiments. Gregory Blass is acknowledged for critical proofreading of the manuscript. This research was supported by the National Institutes of Health grant HL108880 and American Diabetes Association grant 1-15-BS-172 (to AS), and the Ben J. Lipps Research Fellowship from the American Society of Nephrology (to DVI).

Animal use and welfare should adhere to the NIH Guide for the Care and Use of Laboratory Animals following protocols reviewed and approved by the Institutional Animal Care and Use Committee (IACUC).
1. Kidney Flush
<ol>
	<li>Use 8 to 12 week old male rat (suggested is a Sprague Dawley strain, however other strains of different age and gender can be used with appropriate changes).</li>
	<li>Anesthetize the animal according to the procedure allowed by IACUC protocol; monitor depth of anesthesia and inspect the animal. The detailed description of the surgery to be performed in 1.3 - 1.8 can be found in Ilatovskaya et al.7</li>
	<li>After proper anesthesia, place the animal on a temperature-controlled surgical table, make a midline incision of the abdomen (up to 3 inches in length), and uncover the vena cava and the aorta.</li>
	<li>Insert ligature around the celiac and superior mesenteric arteries and the abdominal aorta above those; do not ligate.</li>
	<li>Blunt dissect the abdominal aorta below the renal arteries, and place two ligatures around it, but do not ligate, then clamp the aorta above the ligatures and tie the lower thread.</li>
	<li>Catheterize the aorta with a polyethylene PE50 tubing (attached to a syringe pump filled with PBS) below the clamp and fix the catheter with the second ligature; remove the clamp, turn on the pump, and ligate the aorta and mesenteric with celiac arteries. Quickly make incision in renal vein to relieve the pressure.</li>
	<li>Infuse the aorta with the pre-chilled PBS for 2 or 3 min at a rate of 6 ml/min.</li>
	<li>Stop perfusion, excise and decapsulate7 the kidneys, and put them on ice in the PBS solution. Euthanize the animal according to protocol approved by IACUC.</li>
</ol>
2. Isolation of the Rat Glomeruli
<ol>
	<li>Prepare 30 ml of fresh solution of 5% BSA in RPMI 1640.</li>
	<li>Using a razor blade and scissors, isolate the cortex of both kidneys, and then mince until homogenous. This procedure has been described earlier7.</li>
	<li>Push the tissue minced during the previous step through a 100 mesh stainless steel sieve (pre-soaked in 5% BSA/RPMI solution) using a spatula. Collect the flow-through and by gravity force allow the flow-through to pass through a 140 mesh sieve.</li>
	<li>Filter the flow-through collected from the 140 mesh sieve using a pre-soaked 200 mesh sieve, discard the filtrate, and wash the top of the sieve with 10 - 15 ml of the prepared BSA/RPMI solution to collect the glomeruli that sediment on the sieve.</li>
	<li>Put the BSA/RPMI solution containing glomeruli on ice in a 15 ml tube and let the glomeruli sediment at the bottom of the tube for up to 20 min. The glomeruli concentrate on the bottom of the tube will be clearly seen. Remove the excess solution, leaving approximately 2 ml in the tube.</li>
</ol>
3. Single-channel Patch-clamp Electrophysiology
<ol>
	<li>Prepare 5 x 5 mm cover glass chips by coating them with MW 70,000 - 150,000 poly-ʟ-lysine, and let dry. Use approximately 30 µl of 0.01% sterile filtered solution in water per cover glass.</li>
	<li>Warm up the experimental solutions to RT and fill the patch-clamp chamber and pipette. For TRPC channels monitoring, use a bath solution, in mM: 126 NaCl, 1 CaCl2, 10 HEPES, 2 MgCl2, 10 glucose, pH 7.4; pipette: 126 NaCl, 1.5 CaCl2, 10 HEPES, 10 glucose; pH 7.4.
	<ol>
		<li>Add inhibitors to the pipette solution to block activity of endogenous channels, which are not relevant for the studies (recommended are: 100 µM niflumic acid or DIDS (to block Ca2+-activated Cl- channels), 10 mM TEA (to inhibit the large-conductance Ca2+-dependent K+ channel), 10 nM iberiotoxin (to block the Ca2+-activated K+ channels), 10 µM nicardipine (to block N-type Ca2+ channels)) directly before the patch-clamp experiment.</li>
	</ol>
	</li>
	<li>Gently mix the glomeruli-containing solution, and then apply approximately 50 µl of it to the poly-ʟ-lysine coated cover glass chips. Let the glomeruli attach for approximately 5 min.</li>
	<li>Move the glass chips with glomeruli to the patch-clamp chamber pre-filled with the bath solution; perfuse the chamber at a rate of 3 ml/min for 1 min to ensure the removal of the unattached glomeruli.</li>
	<li>Conduct a conventional patch-clamp experiment in a cell-attached mode7. With a glass pipette (7 - 10 MΩ pipette resistance) form a high-resistance seal between a pipette and a podocyte membrane by applying gentle suction (a pipette attached to a podocyte on the surface of the isolated glomerulus is shown in Figure 2, on the left).</li>
	<li>For the cell-attached measurements, low-pass the currents at 300 Hz by an eight-pole Bessel filter.</li>
	<li>Use isolated glomeruli in patch-clamp experiments for up to 4 - 6 hr. Keep the stock glomeruli fraction on ice.</li>
</ol>
4. Ratiometric Confocal Fluorescence Measurements of Intracellular Calcium Concentration in the Podocytes
<ol>
	<li>Place 500 µl of the glomeruli fraction (described at 2.5) in 0.5 ml conical tube and add calcium dyes Fura Red, AM and Fluo-4, AM. Use 2 mM and 1 mM stock concentrations of Fura Red, AM and Fluo-4, AM, respectively (store at -20 °C, dissolved in DMSO) and use 2.5 µl of each dye for a 500 µl of glomeruli fraction. Immediately after addition of the dyes cover the tube with aluminum foil.</li>
	<li>Place tubes on a rotating shaker for at least 20 min up to 1 hr at RT. 
	Note: Pharmacological agents can be added during this step.</li>
	<li>Prepare glass coverslips, cover them with poly-ʟ-lysine and allow drying using heated plate set to 70 °C.</li>
	<li>Once the loading of calcium dyes is complete, apply 100 µl of the glomeruli containing solution to the poly-ʟ-lysine coated coverslips and let them stick to the surface for about 5 min. Mount the glomeruli-attached coverslips into an imaging chamber, and perfuse with the bath solution (containing (in mM): 145 NaCl, 2 CaCl2, 4.5 KCl, 2 MgCl2, 10 HEPES, pH 7.35) at a rate of 3 ml/min to remove the unattached glomeruli and the remaining dyes.</li>
	<li>Set the confocal laser scanning microscope to a 488 nm excitation wavelength and emission filters (525/25 and 650/25 nm for Fluo-4 and Fura Red, respectively). Set the imaging software to a desired frequency and resolution.</li>
	<li>Find the glomeruli in brightfield and then turn on the detection of the fluorescence signal. Adjust the intensity of the laser for each dye to avoid saturation of the signal. Choose the focal plane with the podocytes which are directly attached to the glass. This minimizes the effect caused by contraction of a glomerulus in response to drug application. Double-check that the glomerulus of choice is well attached to the glass;</li>
	<li>Start imaging the focal plane of choice (take 512 x 512 images with the frequency set at 4 sec to visualize fast Ca2+ transient changes. Use a 60X/NA 1.4 or similar objective lens for high resolution image), apply drugs of interest, and record the response.
	<ol>
		<li>Select a desired focal plane (as close to the surface of the glass as possible, to ensure imaging of podocytes on the surface of the glomerulus). Check the intensity of fluorescence on the Fluo4 and FuraRed channels, and make sure that the glomerulus is clearly seen in brightfield.</li>
		<li>Start imaging. Before application of any drugs, record at least 1 min of baseline fluorescence to make sure that the signal is stable (there are no sudden spikes or fading of the signal).</li>
		<li>Apply desired drugs with the help of a micropipette; be careful and ensure the drug was able to diffuse well and reach the glomerulus. Mix the bath solution gently if needed, monitor the selected focal plane and check that it did not move out of focus because of the drug application.</li>
		<li>Record the changes in fluorescence intensity for the Fluo4 and FuraRed signals. Make sure the recording is long enough, by waiting until the signal reaches the plateau level or reaches a peak and then returns to baseline. Perform solution change or addition of any other drugs if needed.</li>
		<li>Stop the recording, and save the file in the native format of the software.</li>
	</ol>
	</li>
</ol>
5. Image Analysis for the Calcium Measurements
<ol>
	<li>Perform image analysis with the ImageJ open software equipped with the ND Utility plugin that allows importing images in the native ND2 format.</li>
	<li>Import the image sequence; make sure to split the channels and use a hyperstack grayscale mode.</li>
	<li>Follow the Analyze → Tools → ROI manager path in the ImageJ software to open a ROI manager window. Select several regions of interest (podocytes) using an oval selection tool and the “Add (t)” function in the ROI Manager. As the last ROI, select the area in the background; save the selected ROIs (More → Save).</li>
	<li>Highlight the window containing the channel selected for analyses. Use the More → Multi Measure function in the ROI Manager, mark the “one row per slice” box in the dialogue that pops out, and then click OK. The results will be shown in the format that can be set up in the Results window: enter into the menu option Results → Set Measurements, select “Mean gray value” and calculate the pixel intensity values for each ROI, which will be displayed in the Results window in separate columns for each ROI.</li>
	<li>Copy the measured ROI intensity values for each channel (Fluo-4 and Fura Red) into preferred data analysis software; subtract background intensity values from each data point.</li>
	<li>For each time point calculate the ratio of intensities of the Fluo-4 to Fura Red channels. Plot scatter/line point-time changes of Ca2+ transient for each ROI. Calculate Mean/SE values for selected glomeruli. 
	Note: Time column should be set according to imaging frequency selected at 4.5.</li>
</ol>
6. Intracellular Calcium Concentration Calculations Using Fluo-4 Fluorescence Signal
<ol>
	<li>Take a sample of the glomeruli and perform the experimental protocol up to step 4.7. After recording of the background fluorescence add Ionomycin (final concentration in the bath chamber should be 10 µM) to the bath solution, and record fluorescence intensity increase. Once the intensity reaches its maximum and the decay starts, add MnCl2 (final concentration should be 5 mM) to quench the fluorescence8 .</li>
	<li>Analyze the data obtained in 6.1. Copy the Fluo-4 signal ROI intensity values obtained according to the protocol described at 5.1 - 5.5 by preferred analysis software.</li>
	<li>Calculate intracellular calcium concentration (in nM) in the podocyte corresponding to the ROI using a formula: 
	<img alt="Calcium ion concentration equation [Ca²⁺] in biochemistry, formula for free ions calculation." src="/files/ftp_upload/52850/52850equation1.jpg" data-altupdatedat="1787630408000" data-alt="Equation 1"> 
	where Kd is a pre-determined dissociation constant for Fluo-4 (345 nM), F is the intensity at the timepoint that you are calculating the calcium concentration for (baseline), and Fmin and Fmax are the intensity values at the point of maximum calcium load (after ionomycin application) and after quenching of the fluorescence (with MnCl2), respectively (see Figure 3).</li>
</ol>

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<li>Schaldecker, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+the+TRPC5+ion+channel+protects+the+kidney+filter%5BTitle%5D)+AND+%22J.+Clin.+Invest%22%5BJournal%5D)">Inhibition of the TRPC5 ion channel protects the kidney filter.</a> J. Clin. Invest. 123, 5298-5309 (2013).</li>
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<li>Anderson, M., Roshanravan, H., Khine, J., Dryer, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Angiotensin+II+activation+of+TRPC6+channels+in+rat+podocytes+requires+generation+of+reactive+oxygen+species%5BTitle%5D)+AND+%22J.+Cell.+Physiol%22%5BJournal%5D)">Angiotensin II activation of TRPC6 channels in rat podocytes requires generation of reactive oxygen species.</a> J. Cell. Physiol. 229, 434-442 (2014).</li>
</ol>

The authors have nothing to disclose.

The approach described here allows for the analysis of calcium handling by the podocytes of the rodent glomeruli. This technique allows application of patch-clamp single channel electrophysiology and fluorescence ratiometric confocal imaging. However, both approaches can be used separately, on their own. The proposed protocol has several relatively simple steps, including 1) kidney flush; 2) isolation of the glomeruli by differential sieving; 3) performing patch-clamp electrophysiological experiments, or incubation of th...

Kidneys maintain homeostatic balance for various substances and regulate blood volume in a way that determines total blood pressure. Disturbances in the renal filtration, reabsorption or secretion lead to or accompany pathological states, ranging from hyper- or hypotension to end stage renal disease that eventually requires kidney transplantation. The renal filtering unit (glomerulus) consists of three layers &#8211; the capillary endothelium, basement membrane and a single-cell layer of epithelial cells &#8211; podocytes, which play a major role in the maintenance of the slit-diaphragm integrity and function1. Dysfunction in the permselective glomerular filter causes urinary loss of macromolecules, such as proteinuria. Various agents may affect the structure of the podocytes and their foot processes, which determine the integrity of the glomeruli filtration barrier.
The podocytes are involved in the maintenance of the glomeruli filtration function. It has been established that improper calcium handling by the podocyte leads to cell injury and plays an important role in the progression of various forms of nephropathies2,3. Therefore, development of a model which allows for direct measuring of intracellular calcium concentration changes will be instrumental for studies of podocyte function. Isolated glomeruli were previously used in a numerous studies including measurement of albumin reflection coefficient changes4 and assessment of integral cellular currents in the whole-cell electrophysiological patch-clamp measurements5,6. In the present paper we describe the protocol that allows the researcher to measure intracellular calcium concentration changes in response to applications of pharmacological agents, estimate basal levels of calcium within the cells, and assess individual calcium channels activity. Ratometric calcium concentration measurements and patch-clamp electrophysiology were used to determine changes in the intracellular calcium concentration within the podocyte and channel activity, respectively.

<table><tbody><tr><td>Fluo4 AM</td><td>Life Technologies</td><td>F14217</td><td>500 &amp;micro;l in DMSO</td></tr><tr><td>FuraRed AM</td><td>Life Technologies</td><td>F-3020</td><td></td></tr><tr><td>Poly-&amp;#671;-lysine</td><td>Sigma-Aldrich</td><td>P4707</td><td></td></tr><tr><td>Pluronic acid</td><td>Sigma-Aldrich</td><td>F-68</td><td>solution</td></tr><tr><td>Ionomycin</td><td>Sigma-Aldrich</td><td>I3909-1ML</td><td></td></tr><tr><td>Tube rotator</td><td>Miltenyi Biotec GmbH</td><td>130-090-753</td><td>Germany</td></tr><tr><td>Nikon confocal microscope (inverted)</td><td>Nikon</td><td>Nikon A1R</td><td>Laser exitation 488 nm. Emission filters 500-550 nm and 570-620 nm</td></tr><tr><td>Objective</td><td>Nikon</td><td>Plan Apo 60x/NA 1.4 Oil</td><td></td></tr><tr><td>Cover Glass</td><td>Thermo Scientific</td><td>6661B52</td><td></td></tr><tr><td>High vacuum grease</td><td>Dow Corning</td><td></td><td>Silicone Compound</td></tr><tr><td>Software</td><td>Nikon</td><td>Nikon NIS-Elements</td><td></td></tr><tr><td>Recording/perfusion chamber</td><td>Warner Instruments</td><td>RC-26</td><td></td></tr><tr><td>Patch clamp amplifier</td><td>Molecular Devices</td><td>MultiClamp 700B</td><td></td></tr><tr><td>Data acquisition system</td><td>Molecular Devices</td><td>Digidata 1440A</td><td>Axon Digidata&amp;reg; System</td></tr><tr><td>Low pass filter</td><td>Warner Instruments</td><td>LPF-8</td><td>8 pole Bessel</td></tr><tr><td>Borosilicate glass capillaries</td><td>World Precision Instruments</td><td>1B150F-4</td><td></td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument Co</td><td>P-97</td><td>Flaming/Brown type micropipette puller</td></tr><tr><td>Microforge</td><td>Narishige</td><td>MF-830</td><td>Japan</td></tr><tr><td>Motorized micromanipulator</td><td>Sutter Instrument Co</td><td>MP-225</td><td></td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclipse Ti</td><td></td></tr><tr><td>Microvibration isolation table</td><td>TMC</td><td></td><td>equipped with Faraday cage</td></tr><tr><td>Multichannel valve perfusion system</td><td>AutoMake Scientific</td><td>Valve Bank II</td><td></td></tr><tr><td>Recording/perfusion chamber</td><td>Warner Instruments</td><td>RC-26</td><td></td></tr><tr><td>Software</td><td>Molecular Devices</td><td>pClamp 10.2</td><td></td></tr><tr><td>Nicardipine</td><td>Sigma-Aldrich</td><td>N7510</td><td></td></tr><tr><td>Iberiotoxin</td><td>Sigma</td><td>I5904-5UG</td><td></td></tr><tr><td>Niflumic acid</td><td>Sigma-Aldrich</td><td>N0630</td><td></td></tr><tr><td>DIDS</td><td>Sigma-Aldrich</td><td>D3514-25MG</td><td></td></tr><tr><td>TEA chloride</td><td>Tocris</td><td>T2265</td><td></td></tr><tr><td>RPMI 1640</td><td>Life Technologies</td><td>11835030</td><td>without antibiotics</td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A8327</td><td>30% albumin solution</td></tr><tr><td>Temperature controlled surgical table</td><td>MCW core</td><td></td><td>for rodents</td></tr><tr><td>Steel sieves:</td><td></td><td></td><td>#100 (150 &amp;mu;m), 140 (106 &amp;mu;m)</td></tr><tr><td>Gilson, Inc SIEVE 3 SS FH NO200</td><td>Fisher Sci</td><td>50-871-316</td><td></td></tr><tr><td>Gilson, Inc SIEVE 3 SS FH NO270</td><td>Fisher Sci</td><td>50-871-318</td><td></td></tr><tr><td>Gilson, Inc SIEVE 3 SS FH NO400</td><td>Fisher Sci</td><td>50-871-320</td><td></td></tr><tr><td>mesh 200</td><td>Sigma-Aldrich</td><td>s4145</td><td>screen for CD-1</td></tr><tr><td>Binocular microscope</td><td>Nikon</td><td>Eclipse TS100</td><td></td></tr><tr><td>Binocular microscope</td><td>Nikon</td><td>SMZ745</td><td></td></tr><tr><td>Syringe pump-based perfusion system</td><td>Harvard Apparatus</td><td></td><td></td></tr><tr><td>Polyethylene tubing</td><td>Sigma-Aldrich</td><td>PE50</td><td></td></tr><tr><td>Isofluorane anesthesia</td><td><a href="http://www.vetequip.com/" target="_blank"><img src="/img/new_window.png"> VetEquip, Inc.</a></td><td>911103</td><td></td></tr><tr><td>Other basic reagents</td><td>Sigma-Aldrich</td><td></td><td></td></tr></tbody></table>

single-channel analysis and calcium imaging in the podocytes of the freshly isolated glomeruli

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.

Here we addressed the problem of measuring acute changes in the calcium levels in the podocytes. Figure 1 shows a schematic representation of the experimental protocol designed in order to perform high resolution live fluorescence confocal imaging and single ion channel activity recordings in the podocytes of the freshly isolated rodent glomeruli. Briefly, after the rat is anaesthetized, the kidneys should be flushed with PBS to clear them of blood. Then, the kidneys are excised and decapsulated, and glo...

Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...

The overall goal of this procedure is to measure the intracellular calcium concentration changes in response to pharmacological agents. Estimate basal levels of calcium and assess individual channel activity in the podocytes as part of the whole freshly isolated glomerulus. This is accomplished by first clearing blood from rat kidneys by flushing via the abdominal aorta.Next, the kidneys are harvested and the kidney cortex is isolated and minced with a razor blade. In the third step, the cortical glomeruli are isolated by differential cing. Afterward, the isolated decapitated glomeruli can either be subjected to electrophysiological experiments or loaded with fluorescent dyes and taken for confocal calcium concentration measurements.Ultimately, these procedures enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents and assess and manipulate calcium conductance at the level of single ion channels. The main advantage of this technique over existing methods is that it allows us to study podocytes as part of the whole isolated glomerulus. Our preparation retains the functional potential of the podocytes.They remain attached to the capillaries and participate in the homeostasis in the environment that actually can conserves the major parts of the glomeruli filtration apparatus. The implication of this technique extend our pharmacological screening In normal and pathological states, the mechanism of calcium handling by the podocytes serve an essential regulatory role in the development and prevention of renal complications in such diseases as diabetic nephropathy, focal segment omero sclerosis or hypertension. It's a great importance to observe the responsiveness of calcium influx in these cells to drugs, which can be easily screened.In this preparation, VLA Hunka will demonstrate how to flush the kidney for the following procedures. I'll show glomeruli isolation and V clamp electrophysiology, and Dr.GaN will guide you through confocal calcium imaging. To begin this procedure, place an anesthetized rat on the temperature controlled surgical table Under the microscope, make a midline incision on the abdomen to uncover the vein CVA and the aorta.Next, insert the ligature around the celiac and superior mesenteric arteries and the abdominal aorta above them. Blunt, dissect the abdominal aorta below the renal arteries. Then place two ligatures around it, but do not ligate.Clamp the aorta above the ligatures and tie the lower thread. After that, catheterize the aorta with a polyethylene tubing that is attached to a syringe pump filled with PBS below the clamp and fix the catheter with a second ligature. Then remove the clamp and ligate them.Me enteric and celiac arteries. Infuse the aorta with pre chilled PBS for three minutes at a rate of six milliliters per minute. At the same time, make an incision in the vein of cava near the renal veins to relieve the pressure.After three minutes, stop the perfusion, excise and encapsulate the kidneys. Place them in PBS solution on ice. Now prepare 30 milliliters of fresh 5%BSA in RPMI 1640 medium.Using a razor blade and scissors, isolate the cortex of both kidneys, then MIT them until homogenous. Next, push the minced tissue through a 100 mesh stainless steel sieve that has been pre soaked in 5%P-S-A-R-P-M-I solution. Collect the flow through and allow it to pass through a 140 mesh siv.Subsequently, filter the collected flow through from the 140 mesh sieve using a pre soaked 200 mesh siv. Discard the filtrate and wash the top sieve with 10 to 15 milliliters of the prepared B-S-A-R-P-M-I solution and collect glomeruli from the top of the sieve. Next place the B-S-A-R-P-M-I solution containing G Glomeruli in a 15 milliliter, two bon ice and let the glomeruli sediment at the bottom of the tube for up to 20 minutes.In this procedure, coat five by five millimeter cover glass chips with molecular weight, 70, 000 to 150, 000 poly lysine and dry them on a heated plate. Next, warm up the experimental solutions to room temperature and fill the patch clamp chamber and pipette Gently mix the glomeruli containing solution. Then apply approximately 50 microliters of it to each polylysine coated cover glass chip.After that, move the glass chips with glomeruli to the patch clamp chamber. Prefilled with the Beth solution. Perfuse the chamber at a rate of three milliliters per minute for one minute to remove any unattached glomeruli before conducting the conventional patch clamp recording in a cell attached mode.Now place 500 microliters of the glomeruli fraction in each 0.5 milliliter conical tube and add calcium dyes pure red am and flu oh 4:00 AM.Next, place the tubes on a rotating shaker for up to one hour at room temperature. Then prepare the glass cover slips with polylysine and allow them to dry on the heated plate at 70 degrees Celsius. Once the loading of calcium dies is complete, apply 100 microliters of the glomeruli containing solution to the polylysine coated cover slips and let them stick to the surface for about five minutes.Then mount the glomeruli attached cover slips onto an imaging chamber and perfuse them with bath solution at a rate of three milliliters per minute to remove the unattached glomeruli and remaining dyes. Afterward, set the confocal laser scanning microscope to a 488 nanometer excitation wavelength. Then set the imaging software to a desired frequency and resolution.Next, find the glomeruli in brightfield. Subsequently turn on the detection of the fluorescence signal. After that, select a desired focal plane and check the intensity of fluorescence on the flu oh four and firo red channels.Make sure that the glomerulus is clearly seen in brightfield. Then record the changes in fluorescence intensity for the flu oh four and furor red signals. To import the image sequence, split the channels and use the hyper stack gray scale mode.In Image J software, open an ROI manager window following the analyze tools, ROI Manager path. Select one or more regions of interest using an oval selection tool and the add T function in the ROI manager. Then select the area in the background and save the selected ROIs.Next, mark the one row per slice box in the dialogue and click okay in the menu options results. Set measurements. Select mean gray value, and calculate the pixel intensity values for each ROI, which will be displayed in the results window in separate columns.For each ROI then copy the measured ROI intensity values for each channel into the preferred data analysis software, and subtract the background intensity values from each data point for each time point. Calculate the ratio of intensities of the flu oh four to URA red channels after that plot, the point time changes of calcium transient for each ROI. This image shows the patch clamp pipette attached to the oxide body on the surface of the rat glomerulus.During an electrophysiological recording, a part of encapsulated glomerulus can be seen in the right lower corner of the image. Here is a representative current trace of the tripy like channels activity from a cell attached patch made on the podocyte to measure the intracellular calcium concentration. Glomeruli are loaded with flu oh 4:00 AM to label intracellular calcium.This video illustrates the effects of CIN and manganese chloride on calcium influx in the glomeruli. This graph quantifies changes in flu oh four signal intensity recorded from a single podocyte in response to CIN and manganese chloride. As expected, CIN produces the maximum of the fluorescence and manganese chloride quenches the dye and results in the lowest fluorescence intensity.Application of these drugs allows the researcher to later define exact calcium concentration within the cell. This video demonstrates the effect of 10 micromole a TP on the calcium concentration in the glomeruli. Note that there is an increase in the green flu O four fluorescence and a drop in red fu red fluorescence, which account for an increase intracellular calcium concentration.This technique offers a unique opportunity to monitor changes in calcium influx at the single ion channel and individual cells level after pharmacological treatments or other manipulations. Thus, this approach represent a very powerful tool considering multiple available genetically modified rodent models. After watching this video, you should have a good understanding of how to perform calcium ation measurements in the podocytes of the, as well as the electrophysiological, which clamp experiments used together all on its own.These techniques provide an in-depth and mechanistic insight into regulation of calcium handling with podocytes in normal and pathological conditions.

single-channel-analysis-calcium-imaging-podocytes-freshly-isolated

Research

JoVE Journal

Biology

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

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.

One-channel Cell-attached Patch-clamp Recording

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

The goal of this protocol is to isolate intact pacemaker regions of the mouse renal pelvis using vibratome sectioning. These sections can then be used for in situ Ca2+ imaging to elucidate Ca2+ transient properties of pacemaker cells and other interstitial cells in vibratome slices.

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning

The presented method enables visualization of fluorescently labeled cellular proteins with expansion microscopy leading to a resolution of 70 nm on a conventional microscope.

Imaging of Podocytic Proteins Nephrin, Actin, and Podocin with Expansion Microscopy

Cell calcium imaging is a versatile methodology to study dynamic signaling of individual cells, on mixed populations in culture or even on awakened animals, based on the expression of calcium-permeable channels/receptors that gives unique functional signatures.

Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This article presents a method for real-time, quantitative monitoring of calcium ion (Ca2+) concentrations in cells using single-cell Ca2+ imaging with the Fura-2/AM dye. This technique enables efficient dye loading and accurate calculation of Ca2+ levels through fluorescence intensity ratios, making it a simple and rapid approach for research applications.

Single-Cell Calcium Imaging for Studying the Activation of Calcium Ion Channels

Learn calcium imaging and single-channel analysis in podocytes using freshly isolated rat glomeruli. This method enables real-time monitoring of intracellular calcium dynamics and ion channel activity for kidney research.

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

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