Name: synthesis and evaluation of a ruthenium-based mitochondrial calcium uptake inhibitor
Uploaded: 2017-10-26T15:00:00.000+00:00
Duration: 7 min 12 s
Description: Watch this Scientific Journal Video about Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor at JoVE.com

This research was supported by Cornell University. This work made use of the Cornell Center for Materials Research Shared Facilities, which are supported through the NSF MRSEC program (Grant DMR-1120296). S.R.N. acknowledges support by an NSF Graduate Research Fellowship (Grant DGE- 1650441) and Dr. Dave Holowka for assistance with the calcium experiments. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors(s) and do not necessarily reflect the views of the National Science Foundation.

NOTE: Concentrated acids and bases are used in this synthesis. Use all appropriate safety practices when performing the reaction including the use of engineering controls (fume hood) and personal protective equipment (PPE) including safety glasses, gloves, lab coat, full length pants, and closed-toe shoes.
1. Preparation of [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5
<ol>
	<li>Synthesis of [Ru(NH3)5Cl]Cl2 12
	<ol>
		<li>Dissolve 1.00 g of RuCl3&#183;nH2O (40% Ru by weight, 4.1 mmol) in 5 mL of H2O. Cool the dark brown solution to 0 &#176;C in an ice bath. Add 11 mL (0.23 mol) of 80% hydrazine hydrate solution in a dropwise manner. The initial reaction will be vigorous with the evolution gas, resulting in a brown solution. Let the resulting solution stir at room temperature for 16 h; the final solution will be dark red. 
		Caution: Hydrazine is acutely toxic and carcinogenic. Additionally, anhydrous forms of this reagent are explosive. As always, use appropriate PPE and fume hoods when handling. Do not concentrate these solutions to dryness.</li>
		<li>To this solution, add approximately 5-10 mL of concentrated HCl to adjust the pH to 2. At this point, the solution will be yellow-brown in color.</li>
		<li>Heat this solution at 105 &#176;C while stirring for 1-2 h. A yellow solid will precipitate out of solution. When no more precipitate visibly forms, remove from heat.</li>
		<li>Allow the reaction mixture to cool to room temperature, and then place in a 0 &#176;C ice bath for 10 min. Collect the yellow solid by vacuum filtration and wash with 5 mL each of ethanol and diethyl ether.</li>
		<li>Completely dissolve the crude product in 15-25 mL of hot water. Chill 10 mL of a concentrated HCl solution in a filter flask by placing it in an ice bath. Filter the yellow solution into the chilled HCl solution to induce precipitation of a pale yellow pure solid. Filter this precipitate and wash with 5 mL each of 0.5 M HCl, ethanol and ether.</li>
		<li>Characterize the compound using IR spectroscopy. Verify purity by the identification of stretching frequencies at 3226 cm-1, 1604 cm-1, 1297 cm-1, and 801 cm-1. A common minor impurity at 2069 cm-1 is assigned to [Ru(NH3)5N2]Cl3.</li>
	</ol>
	</li>
	<li>Synthesis of [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5
	<ol>
		<li>Dissolve 100 mg (0.34 mmol) [Ru(NH3)5Cl]Cl2 in 50 mL of 1 M NH4OH in a 200 mL heavy wall round-bottomed pressure vessel. Loosely cap the flask with a stopper and heat the reaction mixture at 75 &#176;C for 6 h. Remove from heat and stir at room temperature for 4 days to yield a dark green solution. 
		Caution! Heating a sealed vessel results in a pressure build-up. Make sure to use appropriate pressure-safe glassware. For this reaction, the purpose of sealing the vessel is to minimize loss of gaseous NH3. Therefore, place the stopper loosely to allow for release of excess pressure.</li>
	</ol>
	</li>
	<li>Purification by cation-exchange chromatography
	<ol>
		<li>In a 25 mL beaker, suspend 5 g cation-exchange resin (e.g., Dowex 50WX2 200-400 mesh (H+ form) in 10 mL 0.1 M HCl.</li>
		<li>Load this slurry into a 10 mL column (10 mm diameter, 15 cm height) affixed with a 50 mL solvent reservoir. Wash the resin with approximately 20-30 mL of 0.1 M HCl, until the eluate is colorless.</li>
		<li>Return to the green reaction solution isolated in step 1.2.1. To this solution, add concentrated HCl to adjust the pH to 2, at which point the solution color changes to brown.</li>
		<li>Load this acidified solution to the cation-exchange resin column prepared in step 1.3.2 by gently pipetting it on top of the resin. Let the eluate completely drain, and continue loading the solution. Repeat this process until the entire solution has been added. The top of the resin will be dark brown/black. The resin will decrease in volume slightly.</li>
		<li>Use glass beads to cover the top of the resin. These will prevent the resin from being disturbed when new solutions are added.</li>
		<li>Elute the column with 20 mL of 1 M HCl.</li>
		<li>Elute the column with an increased HCl concentration of 1.5 M (&#8776; 50 mL). A yellow solution will begin to come off the column. Increase the HCl concentration to 2 M and continue eluting until the eluate is colorless or a very pale green-yellow. A total volume of 150-200 mL will be required for this process.</li>
		<li>Increase the HCl concentration to 2.5 M (20-50 mL). Collect the eluate as fractions in test tubes. Increase to 3 M HCl. The product will elute from the column as a green-brown solution. A red-brown fraction may also begin to come off of the column. As these fractions are oxidized ruthenium red impurities, do not pool with the green-brown fractions.</li>
	</ol>
	</li>
	<li>Characterization and verification of purity of [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5
	<ol>
		<li>Test all of the fractions from step 1.3.8. by UV-vis spectroscopy. To accomplish this task, add 100 &#181;L of a given fraction into 2 mL of 3 M NH3 and analyze by UV-vis spectroscopy. Fractions containing pure product will have a large absorbance band at 360 nm and a less intense absorbance at 600 nm. Absorbance at 480 or 533 nm is indicative of oxidized ruthenium red and ruthenium red impurities, respectively.</li>
		<li>Pool fractions containing pure product and evaporate the solution to dryness by rotary evaporation. The product will be isolated as a green-brown solid. Yields are typically on the order of 5-15 mg (10-20% yield). Single-crystals, suitable for X-ray diffraction, can be obtained by the vapor diffusion of ethanol into aqueous solutions of the compound.</li>
		<li>To verify purity, analyze the compound by UV-vis spectroscopy in a solution of pH 7.4 phosphate-buffered saline (PBS). Purity may be assessed by taking the ratio of intensity of the 360 nm and 600 nm peaks. This ratio is 31 for a pure compound. For impure compounds, the ratio will be smaller.</li>
		<li>Analyze the sample in the solid-state by IR spectroscopy. Diagnostic bands are at 3234 cm-1, 3151 cm-1, 1618 cm-1, 1313 cm-1, and 815 cm-1. Common impurity bands are seen at 1762 cm-1 and 1400 cm-1, characteristic of NH4Cl. Ruthenium red can be identified by bands at 1404 cm-1, 1300 cm-1, 1037 cm-1 and 800 cm-1.</li>
	</ol>
	</li>
	<li>Evaluation of mitochondrial calcium uptake inhibition by fluorescence spectroscopy 
	Caution! The following procedures use mammalian cells. Work should be carried out in appropriate laminar flow hoods that are certified for biological safety level 2 (BSL2) research. 
	&#8203;NOTE: [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5 will be referred to as [Ru] in this section
	<ol>
		<li>Make buffered glucose-containing saline solution (BGSS) as the assay media. BGSS is a solution comprising 110 mM KCl, 1 mM KH2PO4, 1 mM MgCl2, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 5 mM sodium succinate, 30 &#181;M ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA). Combine everything except the EGTA, adjust pH to 7.4. Add EGTA and readjust pH to 7.4. For 50 mL of assay media add 0.5 mL of 1 mg/mL glucose.</li>
		<li>Culture HeLa cells in 500 cm2 Petri dishes in Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) in a humidified incubator with 5% CO2 at 37 &#176;C. Amplify HeLa cells growing in a 100 mm Petri dish by seeding them in a 500 cm2 petri dish. The total media volume in the large dish is 115 mL. Each large dish will yield approximately 18 million cells, enough for two fluorescence spectroscopy experiments.
		<ol>
			<li>Grow the cells until they reach 90-95% confluency. Remove media, and rinse the cells with 15 mL pH 7.4 PBS. Add 15 mL of 1 mM ethylenediaminetetraacetic acid (EDTA) in PBS and incubate for 10 min to detach the cells. Transfer the cells to 14 mL round bottom falcon tubes</li>
		</ol>
		</li>
		<li>Count cells using trypan blue and a hemocytometer with an inverted microscope, and calculate the total number of cells and the volume of media needed to reach 7.5 million cells per 1.8 mL volume of medium. Centrifuge the cells for 10 min at 5310 &#215; g. Decant the supernatant and add the calculated volume of BGSS. Resuspend cells gently.
		<ol>
			<li>For this assay, prepare stock solutions of 40 mM digitonin in dimethyl sulfoxide (DMSO), 1 mM Calcium Green-5N in H2O, and 10 mM CaCl2 in H2O. [Ru] stock solutions, prepared in pure water, can range from 1-3 mM. 
			&#8203;NOTE: Calcium Green-5N is light-sensitive. Store in the dark and minimize light exposure.</li>
		</ol>
		</li>
		<li>Setup the fluorimeter to excite at 506 nm and read the emission at 532 nm with the cuvette-holder controlled at 37 &#176;C. Prepare an acrylic cuvette with a stir bar or wheel, 1.8 mL cell suspension from 1.5.2 above, 1.8 &#181;L digitonin solution, 3.6 &#181;L Calcium Green-5N (solution), and 9 &#181;L [Ru] (for 1 mM stock solution, 5 &#181;M final concentration). Incubate cells for 15 min in the fluorimeter.
		<ol>
			<li>Read the data as the excitation/emission ratio instead of the raw absorption. This practice minimizes errors associated with fluctuations in the light source intensity.</li>
			<li>Carry out the first sample analysis in the absence of [Ru] to measure the effect of the addition of CaCl2 on the response of the cells.</li>
			<li>Begin analysis on the fluorimeter with the settings described in 1.5.4. Wait approximately 2 minutes to establish a stable emission baseline, and then add 1.8 &#181;L of CaCl2 (10 &#181;M final concentration). The emission intensity will increase immediately upon the addition of the CaCl2, and will then decay over the course of minutes as the calcium ions enter the mitochondria. Wait until the decay has finished (&#8776;5 min). Add additional calcium boluses to determine the mitochondrial calcium uptake response of cells not treated with [Ru].</li>
		</ol>
		</li>
		<li>In another cuvette containing 5 &#181;M [Ru], repeat the experiment as described above in 1.5.4.3. In the presence of the inhibitor, emission intensity will increase, but not decay. This observation signifies blocked mitochondrial calcium uptake.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose

The mitochondrial calcium uptake inhibitor [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5 can be synthesized from [Ru(NH3)5Cl]Cl2, a well known ruthenium(III) starting material, as described in this procedure. The synthesis of [Ru(NH3)5Cl]Cl2 is readily achieved with little difficulty. After stirring RuCl3 for 16 h in hydrazine hydrate, the pH of the solution should be adjusted to a value of 2 with HCl. The pH drop is critical to achieve the desired product. If desired, this synthesis can be modified to form [Ru(NH3)5Br]Br2 by refluxing in HBr instead of HCl.
We have observed some strategies to minimize formation of undesired byproducts during the synthesis of the mitochondrial calcium uptake inhibitor. Notably, keeping the flask capped is critical to the success of this reaction; if the flask is open, a number of other byproducts, including ruthenium red (RuRed), are formed in appreciable quantities. Presumably, gaseous ammonia is lost from the open reaction vessel, and this event compromises formation of the desired product. The size of the flask and the volume of aqueous ammonia are also important parameters that should not be modified significantly from those described in this protocol, as we have noticed that the efficiency of this reaction is decreased in smaller flasks. This method does not drastically increase the yield of this compound in comparison to the other two methods we had previously disclosed. It does, however, contain fewer steps and give rise to less side product formation, such as RuRed. The low yield may be attributed to the presence of a small amount of unreacted [Ru(NH3)5Cl]Cl2 as well as unknown highly charged impurities that are retained at the top of the column. Although we have not explored many additional reaction conditions, it is possible that improvements can be realized by varying the reaction time and the reactant concentrations, to obtain higher yields.
The purification of [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5 is the most tedious and difficult portion of this protocol. A well packed column will greatly assist in the separation; loading the resin as slurry is an effective approach to pack the column. It should be noted that as the HCl concentration is increased over the course of the column elution, the resin will contract in size. To successfully obtain pure material, the rate at which the acid concentration is increased should not deviate from the stated procedure. The final product will be a green-brown solid; a basic solution will be dark green but acidic solutions will be brown. If a bright pink-red solution is observed, ruthenium red contaminations are present. The amount of RuRed can be determined by using the extinction coefficient (62,000 M-1&#183;cm-1 at 533 nm). This purification process is relatively general for strong cation-exchange resin columns.
The mitochondrial calcium uptake inhibition can be tested using permeabilized HeLa cells, the fluorescent calcium sensor Calcium Green-5N, and a spectrofluorimeter.13,14 This assay requires a large quantity of cells and therefore necessitate amplification of the culture in a very large 500 cm2 Petri dish. These large culture flasks prevent additional challenges in minimizing microbial contamination because the exposed surface areas are very large. Work should be carried out in a laminar flow cabinet to maintain sterility. The fluorescence response of Calcium Green-5N in permeabilized HeLa cells is monitored by fluorescence spectroscopy. The addition of an external bolus of calcium to the cuvette triggers an immediate increase in fluorescence, arising from the calcium ions interacting with the sensor. Over the course of several minutes in the absence of an inhibitor, the intensity decays due to uptake of these calcium ions in the mitochondria, an organelle that cannot be accessed by the dye. When this assay is carried out in the presence of an inhibitor the addition of a bolus of calcium ions gives rise to an increase in emission intensity. The decay due to mitochondrial calcium uptake, however, is not present, verifying the mitochondrial calcium uptake inhibitory properties of this compound. This process can be used as a general screen for calcium uptake inhibitors. In addition, this procedure can be applied to other eukaryotic cells of interest. This assay depends on the permeabilization of the outer cellular membrane thus does not provide information about the cellular uptake ability of a complex.
In summary, this protocol describe the synthesis and purification of a novel ruthenium-based mitochondrial calcium uptake inhibitor. This compound is of significant value for studying the biology of mitochondrial calcium and its role in the physiology of the mammalian cell. The relatively simple assay for testing mitochondrial calcium uptake is also of use for the screening and investigation of new inhibitors.

Mitochondrial calcium is a key regulator for a number of processes that are critical to normal cell function, including energy production and apoptosis.1,2,3 The mitochondrial calcium uniporter (MCU), an ion transporter protein that resides on the inner mitochondrial membrane, regulates the influx of calcium ions into the mitochondria.4,5,6 Chemical inhibitors of the MCU are valuable tools for continuing efforts to study the function and cellular roles of this transport protein and mitochondrial calcium. The compound [(HCO2)(NH3)4Ru(&#181;-O)Ru(NH3)4(O2CH)]3+, Ru360, is one of the only known selective inhibitors for the MCU with a reported Kd value of 24 &#181;M.7,8,9,10 This complex is a common impurity found in commercial formulations of ruthenium red (RuRed), a triruthenium di-&#181;-oxo bridged hexacation of the formula [(NH3)5Ru(&#181;-O)Ru(NH3)4(&#181;-O)Ru(NH3)5)]6+, which has also been used as a calcium uptake inhibitor. Although Ru360 is commercially available, it is very costly. Moreover, the synthesis and isolation of Ru360 is challenged by difficult purification procedures and ambiguous characterization methods.
We have recently reported alternative procedures to access a Ru360 analogue, [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5.11 This compound inhibits the MCU with high affinity, similar to Ru360. In this protocol, we will describe our most effective synthesis of this compound, which commences from [Ru(NH3)5Cl]Cl2. Purification of the product using strongly acidic cation-exchange resin is detailed, along with common pitfalls for this procedure. We also present methods for characterization and assessment of compound purity, and delineate a simple approach to test its efficacy in blocking mitochondrial calcium uptake.

<table><tbody><tr><td>Ruthenium Trichloride hydrate</td><td>Pressure Chemical</td><td>3750</td><td></td></tr><tr><td>Concentrated hydrochloric acid</td><td>J.T. Baker</td><td>9535</td><td></td></tr><tr><td>Concentrated ammonium hydroxide</td><td>Mallinckrodt Chemical Works</td><td>A669C-2 1</td><td></td></tr><tr><td>Dowex 50 WX2 200-400 Mesh</td><td>Alfa Aesar</td><td>13945</td><td></td></tr><tr><td>Calcium Green 5N</td><td>Invitrogen</td><td>C3737</td><td></td></tr><tr><td>Digitonin</td><td>Aldrich</td><td>260746</td><td></td></tr><tr><td>DMSO</td><td>Aldrich</td><td>471267</td><td></td></tr><tr><td>EGTA</td><td>Aldrich</td><td>E3889</td><td></td></tr><tr><td>KCl</td><td>USB</td><td>20598</td><td></td></tr><tr><td>KH2PO4</td><td>Aldrich</td><td>P3786</td><td></td></tr><tr><td>MgCl2</td><td>Fisher Scientific</td><td>M33-500</td><td></td></tr><tr><td>HEPES</td><td>Fluka</td><td>54466</td><td></td></tr><tr><td>Sodium Succinate</td><td>Alfa Aesar</td><td>33386</td><td></td></tr><tr><td>EDTA</td><td>J.T. Baker</td><td>8993-01</td><td></td></tr><tr><td>Glucose</td><td>Aldrich</td><td>G5000</td><td></td></tr><tr><td>200 Round bottom flask</td><td>ChemGlass</td><td>CG-1506-14</td><td></td></tr><tr><td>Glass stopper</td><td>ChemGlass</td><td>CG-3000-05</td><td></td></tr><tr><td>10 mm x 15 cm glass column with reservoirs</td><td>Custom - similar to Chemglass columns</td><td>Similar to CG-1203-20</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10-017-CV</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>10437028</td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Round bottom Falcon tubes</td><td>Fisher Scientific</td><td>14-959-11B&amp;nbsp;</td><td></td></tr><tr><td>500 cm&lt;sup&gt;2&lt;/sup&gt; petri dishes</td><td>Corning</td><td>431110</td><td></td></tr><tr><td>Trypan blue</td><td>ThermoFisher Scientific</td><td>15250061</td><td></td></tr><tr><td>Hemacytometer</td><td>Aldrich</td><td>Z359629</td><td></td></tr><tr><td>Acrylic Cuvettes</td><td>VWR&amp;nbsp;</td><td>58017-875</td><td></td></tr><tr><td>UV-Vis spectrometer</td><td>Agilent Model Cary 8454&amp;nbsp;</td><td></td><td></td></tr><tr><td>Spectrofluorimeter</td><td>SLM Model 8100C</td><td></td><td></td></tr><tr><td>IR spectrometer</td><td>Bruker Hyprion FTIR with ATR attachment</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>ALC Model PM140R</td><td></td><td></td></tr><tr><td>Inverted light microscope</td><td>VWR&amp;nbsp;</td><td>89404-462</td><td></td></tr></tbody></table>

synthesis and evaluation of a ruthenium-based mitochondrial calcium uptake inhibitor

We detail the synthesis and purification of a mitochondrial calcium uptake inhibitor, [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]5+. The optimized synthesis of this compound commences from [Ru(NH3)5Cl]Cl2 in 1 M NH4OH in a closed container, yielding a green solution. Purification is accomplished with cation-exchange chromatography. This compound is characterized and verified to be pure by UV-vis and IR spectroscopy. The mitochondrial calcium uptake inhibitory properties are assessed in permeabilized HeLa cells by fluorescence spectroscopy.

This method describes a synthesis of the mitochondrial calcium uptake inhibitor [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5 starting from [Ru(NH3)5Cl]Cl2, a well known ruthenium(III) starting material. [Ru(NH3)5Cl]Cl2 is characterized by IR spectroscopy, with vibrational modes at 3200 cm-1, 1608 cm-1, 1298 cm-1, and 798 cm-1 (Figure 1). A minor impurity at 2069 cm-1 can be attributed to [Ru(NH3)5N2]Cl3. The reaction of this Ru(III) species with 1 M NH4OH affords [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5. The progress of this reaction is evidenced by a dramatic change in color of the solution from yellow to green. The final reaction solution is dark green in color. Acidification of this solution with concentrated HCl results in a color change to brown; a byproduct of this neutralization is ammonium chloride, which can contaminate the final product if care is not exercised. Purification of the compound proceeds via cation-exchange chromatography using strongly acidic mesh (H+ form) resin. The resin is equilibrated first with 0.10 M HCl, and the solution of [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5 is loaded on the column. The ammonium chloride byproduct elutes with the 1 M HCl washings. Ruthenium-containing compounds elute at higher HCl concentrations. A series of yellow fractions, which contain unreacted [Ru(NH3)5Cl]Cl2 starting material, come off of the column when the HCl concentration is 1.5 M. The desired product elutes between 2.5-3.0 M HCl, and the resulting fractions appear green to green-brown in color.
Prior to pooling the fractions of the product, their purity should be verified. Because the desired compound exhibits some pH-dependence in its UV-vis spectrum, we suggest adding small aliquots of the fractions to highly basic 3 M NH3 solutions to ensure that the spectral features are the same for all of the fractions. Pure fractions should only display an intense peak at 360 nm and a weak peak at 600 nm (Figure 2). Peaks near 480 and 533 nm indicate the presence of ruthenium brown and red, respectively, and a significant peak at 260 nm, with a shoulder at 290 nm, signifies the presence of [Ru(NH3)5Cl]Cl2 starting material (Figure 3). Rotary evaporation of pure fractions affords the desired compound as a green-brown solid.
The isolated compound can further be characterized by both UV-vis and IR spectroscopy. The UV-vis spectrum (Figure 2) displays the 360 nm and 600 nm absorbance bands as described above. The extinction coefficient for the 600 nm band is 850 M-1cm-1 and for the 360 nm band is 27000 M-1cm-1. The ratio of the intensity of the 360 nm band compared to the 600 nm band should be 31 in pH 7.4 PBS, and this metric can effectively be used to gauge the purity of the compound. The IR spectrum should appear as shown in Figure 4. The Ru-O-Ru stretching frequency, for example, is diagnostic at 850 cm-1. The IR spectrum was employed to determine the Ru-O-Ru stretch and to ensure no ammonium chloride was present in the final product. Ammonium chloride, a common impurity, has vibrational modes at 1762 cm-1 (very weak) and 1400 cm-1 (strong) that can be easily discerned from the IR spectrum (Figure 5). Ruthenium red is a common byproduct of the reaction and can be identified in the IR spectrum with stretches at 1404 cm-1, 1300 cm-1, 1037 cm-1 and 800 cm-1, although some overlap with the desired product will occur (Figure 6).
The calcium uptake response in digitonin-permeabilized HeLa cells is observed (Figure 7). The asterisks indicate the addition of a CaCl2 bolus. In the presence of [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5 an increase in emission intensity is observed upon calcium addition, but no decay due to mitochondrial calcium uptake is observed.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56527/56527fig1.jpg" /> 
Figure 1: Infrared spectra of [Ru(NH3)5Cl]Cl2. The infrared spectra of [Ru(NH3)5Cl]Cl2 with a very small [Ru(NH3)5N2]Cl3 impurity. The red arrow indicates the impurity at 2,069 cm-1.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56527/56527fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56527/56527fig2.jpg" /> 
Figure 2: Representative UV-vis spectra for pure material. [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5 UV-vis absorbance spectra and near infrared (inset) taken in pH 7.4 PBS. The extinction coefficient for the major absorbance at 360 nm is 27,000 M-1 cm-1.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56527/56527fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56527/56527fig3.jpg" /> 
Figure 3: UV-vis spectra of the crude reaction mixture. [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5 UV-vis absorbance spectra of the crude reaction mixture before purification taken in pH 7.4 PBS. The red arrows indicate common impurities.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56527/56527fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56527/56527fig4.jpg" /> 
Figure 4: Representative infrared spectra for pure material. The infrared spectra of [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5. The Ru-O-Ru stretch is at 850 cm-1, the other stretches are from the NH3 oscillators.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56527/56527fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56527/56527fig5.jpg" /> 
Figure 5: Infrared spectra containing NH4Cl impurities. The infrared spectra of [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5 with ammonium chloride impurity. The red arrow indicates ammonium chloride at 1,400 cm-1.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56527/56527fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56527/56527fig6.jpg" /> 
Figure 6: Infrared spectra of commercial ruthenium red. The infrared spectrum of [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5 (black trace) and commercial ruthenium red (red trace).&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56527/56527fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56527/56527fig7.jpg" /> 
Figure 7: Representative calcium uptake results. The fluorescence increase due to the addition of calcium to a cocktail of digitonin-permeabilized HeLa cells, Calcium Green-5N and [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5 in BGSS. The blank contains no [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5 and fluorescence decrease due to Ca2+ uptake into the mitochondria can be seen.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56527/56527fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor at JoVE.com

Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor

A protocol for the synthesis, purification, and characterization of a ruthenium-based inhibitor of mitochondrial calcium uptake is presented. A procedure to evaluate its efficacy in permeabilized mammalian cells is demonstrated.

We detail the synthesis and purification of a mitochondrial calcium uptake inhibitor, [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]5+. The optimized synthesis of ...

synthesis-evaluation-ruthenium-based-mitochondrial-calcium-uptake

Nanyang Technological University

Research

JoVE Journal

Chemistry

7.8K Views.  Cornell University. Een protocol voor de synthese, zuivering en karakterisering van een ruthenium gebaseerde remmer van mitochondriale calcium opname wordt gepresenteerd. Een procedure voor de evaluatie van de werkzaamheid bij zoogdiercellen permeabel is aangetoond.

Video: Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the hydroxylmethylene phosphine precursor with ammonia in methanol under a nitrogen atmosphere. The N-triphosPh ligand precipitates from the solution after approximately 1 hr of reflux and can be isolated analytically pure via simple cannula filtration procedure under nitrogen. Reaction of the N-triphosPh ligand with [Ru3(CO)12] under reflux affords a deep red solution that show evolution of CO gas on ligand complexation. Orange crystals of the complex [Ru(CO)2{N(CH2PPh2)3}-&#954;3P] (2) were isolated on cooling to RT. The 31P{1H} NMR spectrum showed a characteristic single peak at lower frequency compared to the free ligand. Reaction of a toluene solution of complex 2 with oxygen resulted in the instantaneous precipitation of the carbonate complex [Ru(CO3)(CO){N(CH2PPh2)3}-&#954;3P] (3) as an air stable orange solid. Subsequent hydrogenation of 3 under 15 bar of hydrogen in a high-pressure reactor gave the dihydride complex [RuH2(CO){N(CH2PPh2)3}-&#954;3P] (4), which was fully characterized by X-ray crystallography and NMR spectroscopy. Complexes 3 and 4 are potentially useful catalyst precursors for a range of hydrogenation reactions, including biomass-derived products such as levulinic acid (LA). Complex 4 was found to cleanly react with LA in the presence of the proton source additive NH4PF6 to give [Ru(CO){N(CH2PPh2)3}-&#954;3P{CH3CO(CH2)2CO2H}-&#954;2O](PF6) (6).

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

Ruthenium phosphine complexes are widely used for homogeneous catalytic reactions such as hydrogenations. The synthesis of a series of novel tridentate ruthenium complexes bearing the N-triphos ligand N(CH2PPh2)3 is reported. Additionally, the stoichiometric reaction of a dihydride Ru&#8211;N-triphos complex with levulinic acid is described.

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the ...

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

Biochemistry

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic macrocycles that exhibit differential cytostatic and cytotoxic properties in specific cell lines, depending on the identities of the chelated metal and functional groups. The unique behavior of functionalized corroles towards specific cell lines introduces the possibility of targeted chemotherapy.
Many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Here we present a step-by-step protocol for RNA transcription in the presence of known and potential inhibitors. The evaluation of the RNA products of the transcription reaction by gel electrophoresis and UV-Vis spectroscopy provides information on inhibitive properties of potential anticancer drug candidates and, with modifications to the assay, more about their mechanism of action.
Little is known about the molecular mechanism of action of corrole cytotoxicity. In this experiment, we consider two corrole compounds: gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc). An RNA transcription assay was used to examine the inhibitive properties of the corroles. Five transcription reactions were prepared: DNA treated with Actinomycin D, triptolide, Ga(tpfc), tpfc at a [complex]:[template DNA base] ratio of 0.01, respectively, and an untreated control.
The transcription reactions were analyzed after 4 hr using agarose gel electrophoresis and UV-Vis spectroscopy. There is clear inhibition by Ga(tpfc), Actinomycin D, and triptolide.
This RNA transcription assay can be modified to provide more mechanistic detail by varying the concentrations of the anticancer complex, DNA, or polymerase enzyme, or by incubating the DNA or polymerase with the complexes prior to RNA transcription; these modifications would differentiate between an inhibition mechanism involving the DNA or the enzyme. Adding the complex after RNA transcription can be used to test whether the complexes degrade or hydrolyze the RNA. This assay can also be used to study additional anticancer candidates.

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

Gallium(III) 5,10,15-(tris)pentafluorophenylcorrole and its freebase analogue exhibit low micromolar cell cytotoxicity. This manuscript describes an RNA transcription reaction, imaging RNA with an ethidium bromide-stained gel, and quantifying RNA with UV-Vis spectroscopy, in order to assess transcription inhibition by corroles and demonstrates a straightforward method of evaluating anticancer candidate properties.

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic ...

Bioengineering

Dose Uptake of Platinum- and Ruthenium-based Compound Exposure in Zebrafish by Inductively Coupled Plasma Mass Spectrometry with Broader Applications

Metals and metal-based compounds comprise multifarious pharmaco-active and toxicological xenobiotics. From heavy metal toxicity to chemotherapeutics, the toxicokinetics of these compounds have both historical and modern-day relevance. Zebrafish have become an attractive model organism in elucidating pharmaco- and toxicokinetics in environmental exposure and clinical translation studies. Although zebrafish studies have the benefit of being higher-throughput than rodent models, there are several significant constraints to the model.
One such limitation is inherent in the waterborne dosing regimen. Water concentrations from these studies cannot be extrapolated to provide reliable internal dosages. Direct measurements of the metal-based compounds allow for a better correlation with compound-related molecular and biological responses. To overcome this limitation for metals and metal-based compounds, a technique was developed to digest zebrafish larval tissue after exposure and quantify metal concentrations within tissue samples by inductively coupled plasma mass spectrometry (ICPMS).
ICPMS methods were used to determine the metal concentrations of platinum (Pt) from cisplatin and ruthenium (Ru) from several novel Ru-based chemotherapeutics in zebrafish tissue. Additionally, this protocol distinguished concentrations of Pt that were sequestered in the chorion of the larval compared with the zebrafish tissue. These results indicate that this method can be applied to quantitate the metal dose present in larval tissues. Further, this method may be adjusted to identify specific metals or metal-based compounds in a broad range of exposure and dosing studies.

The increased rate of pharmaco- and toxicokinetic analyses of metals and metal-based compounds in zebrafish can be advantageous for environmental and clinical translation studies. The limitation of unknown waterborne exposure uptake was overcome by conducting trace metal analysis on digested zebrafish tissue using inductively coupled plasma mass spectrometry.

Metals and metal-based compounds comprise multifarious pharmaco-active and toxicological xenobiotics. From heavy metal toxicity to chemotherapeutics, the ...

Changing Mitochondrial Function for Therapeutic and Bioinsecticide Applications

Unveiling Xenobiotic Transport and Effects in Isolated Mitochondria: Insights from Respirometric and Enzymatic Assays

Mitochondria are often referred to as the cell's powerhouse due to their crucial role in energy production through the electron transport chain (ETC). However, their significance extends far beyond energy production. Dysregulation of mitochondrial bioenergetics can trigger intracellular cascades that impact health and cellular function. Given their physiological importance, there is growing interest in exploring the pharmacological potential of mitochondrial function for developing new therapies and bioproducts. Modulating mitochondrial bioenergetics could provide innovative approaches to address challenges such as neurodegenerative disorders, metabolic diseases, and cancer, as well as to develop bioproducts for controlling pests and vector-borne diseases.
This work presents a method for assessing the effects of xenobiotics on the electron transport chain (ETC) using various substrates, including glutamate and NADH for complex I, succinate for complex II, and cytochrome c (both oxidized and reduced) for complexes III and IV. This methodology allows the activation or inhibition of electron transport through mitochondrial complexes to be evaluated using a respirometer and spectrophotometer. The mitochondria can be sourced from isolated mitochondria, fragmented cells, or homogenized tissue from various species.
In the laboratory, mitochondrial function has been analyzed in Aedes aegypti and Wistar rats, but this method is also applicable to other species, such as Rhipicephalus microplus and Rhodnius prolixus. This approach provides the basis for theorizing about the existence of uncoupler proteins and species-specific oxidizable substrate preferences influenced by their unique energetic demands. Recent findings offer valuable insights into innovative bioinsecticide design strategies that target mitochondrial function, holding significant potential for effectively controlling vector-borne diseases and pest infestations.

This study introduces a methodology for studying xenobiotic effects on mitochondrial function via respirometric and enzymatic assays. It examines electron transport chain modulation in various species, offering insights for targeted biopesticide development against insect vector-borne diseases.

Mitochondria are often referred to as the cell's powerhouse due to their crucial role in energy production through the electron transport chain (ETC). ...

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in cytosolic Ca2+ buffering, and the ATP production in mitochondrial can be mediated by intramitochondrial free Ca2+ concentration. Ca2+ retention capacity can be regarded as the capability of mitochondria to retain calcium in the mitochondrial matrix. Accumulated intracellular Ca2+ leads to the permeability of the inner mitochondrial membrane, termed the opening of mitochondrial permeability transition pore (mPTP), which leads to the leakage of molecules with a molecular weight less than 1.5 kDa. Ca2+-triggered mitochondria swelling is used to indicate the mPTP opening. Here, we describe two assays to examine the Ca2+ retention capacity and Ca2+-triggered mitochondrial swelling in isolated mitochondria. After certain amounts of Ca2+ are added, all steps can be completed in one day and recorded by a microplate reader. Thus, these two simple and effective assays can be adopted to assess the Ca2+-related mitochondrial functions.

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

This protocol aims to describe a method to examine the Ca2+ retention capacity and Ca2+- triggered mitochondrial swelling of isolated mitochondria of SH-SY5Y cells step-by-step.

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in ...

Neuroscience

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Biology

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay

Spirocyclic heterocycles have recently been reported in literature to be potential drugs for cancer therapy. The synthesis of these novel orthogonal ring systems is challenging. An efficient methodology to synthesize these compounds was recently published that described the solid phase synthesis in four steps rather than the previously reported five steps. The advantage of this shorter synthesis is the elimination of the use of toxic reagents. Low-loading Regenerating Michael (REM) linker-based resin was found to be crucial in the synthesis as high-loading versions prevented the addition of reagents containing bulky phenyl and aromatic side chains. The colorimetric 3-(4&#8242;,5&#8242;-dimethylthiazol-2&#8242;-yl)-2,5- diphenyltetrazolium bromide (MTT) assay was used to examine the cytotoxicity of micromolar concentrations of these novel spirocyclic molecules in vitro. MTT is readily available commercially and produces relatively fast, reliable results, making this assay ideal for these spirocyclic heterocycles. Orthogonal ring structures as well as furfurylamine (a precursor in the synthesis method containing a similar 5-member ring motif) were tested.

Here, we describe a bioassay using 3-(4&#8242;,5&#8242;-dimethylthiazol-2&#8242;-yl)-2,5- diphenyltetrazolium bromide (MTT) to test previously synthesized spirocyclic oximes.

Spirocyclic heterocycles have recently been reported in literature to be potential drugs for cancer therapy. The synthesis of these novel orthogonal ring ...

Calcium Influx Assay to Measure Mitochondrial Calcium Uptake in Cultured Cells

Source: Maxwell, J.T., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57225">Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells.</a> J. Vis. Exp. (2018).
This video describes a confocal microscopy imaging-based assay to measure mitochondrial calcium uptake in cultured cells. The assay uses the calcium-sensitive fluorescent dye Rhod-2/AM to measure the mitochondrial calcium in permeabilized cultured cells.

Source: Maxwell, J.T., et al., Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells. J. Vis. Exp. (2018).
This video ...

Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor

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