This work was partially supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2018R1A6A3A01011832), by the Ministry of Science, ICT &#38; Future Planning (NRF-2016M3C1A6936606) and by the Ministry of Trade, Industry &#38; Energy (10068076).

All experimental protocols were approved by the local institutional animal care and use committee.
1. Solution preparation
<ol>
	<li>Prepare single freshly isolated cardiac myocytes11. 
	NOTE: Each laboratory might have a different cell storage solution. Here, the myocytes are stored in culture medium (DMEM).</li>
	<li>Prepare 100 mL of Ca2+-free solution (Table 1).</li>
	<li>Prepare 50 mL of culture medium in a 50 mL beaker. Aliquot 5 mL and put it in a water bath at 37 &#176;C. Keep the remaining solution at room temperature.</li>
	<li>Prepare 50 mL of the saponin solution by adding 5 mg of saponin to 50 mL of Ca2+-free solution. 
	NOTE: Saponin is used to permeabilize cardiac myocytes, to remove cytosolic compartments, and to visualize the mitochondrial fluorescence only.</li>
	<li>Prepare 16 &#956;L of 1 mM fura-2-FF-AM dissolved in dimethyl sulfoxide (DMSO). 
	NOTE: Make 1 mM stock solution of fura-2-FF-AM dissolved in DMSO and aliquot 16 &#956;L in a 2 mL tube. Store them at -20 &#176;C until use.</li>
	<li>Prepare 50 mL of NADH-free Ca2+-free solution (Table 1) and 50 mL of NADH-free Ca2+-saturated solution (Table 1) when isosbestic points are to be measured. Adjust pH to 7.0 with KOH. 
	NOTE: NADH-free Ca2+-free solution (Table 1) contains 10 &#181;M FCCP and 100 &#181;M ADP without any mitochondrial substrates to minimize NADH in mitochondria.</li>
	<li>Prepare 50 mL of Ca2+-free solution, 50 mL of malate solution, 50 mL of pyruvate solution, 50 mL of malate-pyruvate solution, and 50 mL of rotenone solution to be used for NADH correction factor measurements (Table 1).</li>
</ol>
2. Fluoroprobe loading procedure into the mitochondria
<ol>
	<li>Prepare the dye-loading solution by adding 2 mL of the culture medium to 16 &#181;L of 1 mM fura-2-FF-AM. 
	NOTE: Fluorescent dye is fragile under the light. Prepare the solution just before use. Keep the solution containing the fluorescent dye in a dark place. The final concentration of fura-2-FF-AM is 8 &#181;M. If carboxy-SNARF-1 was used, prepare the dye loading solution with 2 &#181;M carboxy-SNARF-1-AM.</li>
	<li>Take 2 mL of the isolated cells and place in a 5 mL test tube in an upright position.</li>
	<li>Wait 15 min for myocytes to sink to the bottom and remove the supernatant. 
	NOTE: The supernatant may contain cell debris. Do not centrifuge the tube to avoid cell damage.</li>
	<li>Add 2 mL of the dye-loading solution.</li>
	<li>Incubate the dye-loading solution with cells for 60 min at 4 &#176;C.</li>
	<li>Then, put the test tube in a 37 &#176;C water bath for 30 min in an upright position.</li>
	<li>Remove the supernatant, and add 4 mL of the prewarmed culture medium of 37 &#176;C. Incubate the cells for 60 min in a 37 &#176;C water bath.</li>
	<li>Finally, remove the supernatant, add 4 mL of culture medium at room temperature and keep the tube at room temperature.</li>
</ol>
3. Introduction of the multiparametric measurement system
NOTE: Figure 1 shows a diagram of the whole system.
<ol>
	<li>For an excitation light source, use a fast monochromator (polychrome II) that can change the light within 3 ms.</li>
	<li>Use an oil immersion lens (40x, NA 1.3) with an inverted microscope to increase the signal intensity.</li>
	<li>Use a near-infrared filter and a charge-coupled device (CCD) camera to monitor the object field without fluorescent signal interference.</li>
	<li>Capture the object field image to get the area.</li>
	<li>Adjust the object field in monitor screen with a field diaphragm just to show the cell for reducing the background.</li>
	<li>Use four photomultiplier tubes with each band-pass filter (450, 500, 590, and 640 nm) to detect emission wavelengths with photon counting method. Use the appropriate dichroic mirrors to split and to redirect the emission light. 
	NOTE: The excitation light is very strong compared to the emission light. Thus, choose the band-pass filter with the highest blocking characteristics to reduce the background. A photon counting system comprises a combination of PMTs, photon counter units, and a high-speed counter. To control the system and to sample the data, a custom-made driving software was used. Finding a way to apply this method with other systems is necessary.</li>
</ol>
4. NADH correction methods with a multiparametric measurement system
<ol>
	<li>Identification of the isosbestic points of Fura-2-FF in situ. 
	NOTE: Many reports have stated that fluorescent characteristics are changed in cells. Therefore, perform all procedures to obtain the parameters to correct the interference in situ.
	<ol>
		<li>Mount the dye-loaded cell on the microscope and wait for 3 min to sink the cells to the bottom. 
		NOTE: Adjust cell numbers to see around one cell per one objective field with 40x objective lens.</li>
		<li>Perfuse the NADH-free Ca2+-free solution at 37 &#176;C.</li>
		<li>After targeting cell, measure the cell-free background and the cell area as shown in section 4.1. Calculate the cell background from the cell area. 
		NOTE: Both background signals need to be corrected in each experiment.</li>
		<li>Perfuse the saponin solution for 60 s and return to the NADH-free Ca2+-free solution.</li>
		<li>Measure Fura-2-FF-emitted signals at 450 nm and 500 nm simultaneously by the excitation scan from 350 nm to 365 nm with the 0.1 nm step.</li>
		<li>Perfuse the NADH-free Ca2+-saturated solution and repeat step 4.2.5.</li>
		<li>Subtract the signals in the Ca2+-saturated solution from the signals in the Ca2+-free conditions.</li>
		<li>Repeat the procedures from 4.2.2 to 4.2.7 with other single cardiac myocytes. 
		NOTE: If the signal intensity become weaker, repeat from 4.2.1. Repeat the procedure for, at least, 5 different cells.</li>
		<li>From all obtained signals, calculate the standard deviations of the emission at each excitation and choose the excitation wavelength showing the minimum standard deviation (SD) value as an isosbestic point. 
		NOTE: The representative figures are shown in Figure 2.</li>
	</ol>
	</li>
	<li>The background signal detection and the correction methods with the cell area 
	NOTE: There are two kinds of the backgrounds. One comes from the cells and the other comes from the reflection on the cover slip (the cell-free background). Both backgrounds need to be corrected in each experiment.
	<ol>
		<li>Mount the dye-free cells in the bath on the microscope and wait for 3 min to sink the cells to the bottom. Perfuse NADH-free Ca2+-free solution for around 5 mins. 
		NOTE: All solution perfusion rate is 2-3 mL/min at 37 &#176;C. 
		NOTE: Adjust cell numbers to see around one cell per one objective field with 40x objective lens.</li>
		<li>Set the object field to cover the targeted cell.</li>
		<li>After moving the cell out of the field, measure the background signals of the cell-free window and set them as offsets. 
		NOTE: The signal means the light signal to be detected in the photon counting system.</li>
		<li>Return the cell to the initial position and measure the cell background signals and the cell area. 
		NOTE: Even though the excitation light is filtered with the bandpass filter, it still contains quite large amount of the filtered light. This light is dispersed when hitting the cells and causes a considerable background signal because the photon counting system is highly sensitive. It needs to be corrected. 
		NOTE: The cell area may be calculated with a captured cell image and an available imaging software. The unit of the cell area can be any unit including pixel count. Just standardization is necessary.</li>
		<li>Repeat from 4.1.1 to 4.1.4 for 10 times to obtain the relationship between the cell area and the cell background signals. 
		NOTE: Later, the cell background signals can be calculated from the cell area from the relationship. Since the excitation light bulb become aging, this procedure needs to be repeated, at least, every month.</li>
	</ol>
	</li>
	<li>Measurement of R factors
	<ol>
		<li>Calculate Rf with the equation 4 from the signals obtained in section 4.2.</li>
		<li>Mount the dye-free cells on the microscope and perfuse the Ca2+-free solution.</li>
		<li>Measure the signals such as F361, 450, NADH, F400, 500, NADH, F361, 450, NADH, and F353, 500, NADH.</li>
		<li>Perfuse the malate solution and repeat 4.3.3. and measure the signals.</li>
		<li>Perfuse the pyruvate solution and repeat 4.3.3. and measure the signals.</li>
		<li>Perfuse the malate-pyruvate solution and repeat 4.3.3. and measure the signals.</li>
		<li>Perfuse the rotenone solution and repeat 4.3.3. and measure the signals. 
		NOTE: The example of the NADH signal recorded on 5 mM pyruvate, 5 mM malate plus 5 mM pyruvate, and 10 &#181;M rotenone addition is shown in Figure 3.</li>
		<li>Calculate each slope of F361, 450, NADH vs. F400, 500, NADH and F361, 450, NADH vs. F353, 500, NADH. As shown in Figure 3. Each slope indicates RN1 and RN2.</li>
	</ol>
	</li>
</ol>
5. Selection of the excitation and the emission light for TMRE or carboxy-SNARF-1
<ol>
	<li>If TMRE for measuring the mitochondrial potential was used in addition, use the 530 nm excitation wavelength and the 590 nm emission wavelength.</li>
	<li>If carboxy-SNARF-1 for measuring the mitochondrial potential was used in addition, use the excitation wavelength of 540 nm and emission wavelengths of 590 nm and 640 nm12.</li>
</ol>
6. Selection of Kd value of fura-2-FF
<ol>
	<li>The change of pH can affect Kd values for Ca2+ binding on fura-2-FF10. Use the Kd value of 5.28 at pH 7.5 for the mitochondria.</li>
</ol>

<ol>
	<li>Berridge, M. J., Bootman, M. D., Roderick, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signalling:+dynamics,+homeostasis+and+remodelling.">Calcium signalling: dynamics, homeostasis and remodelling.</a> Nature Reviews Molecular Cell Biology. 4 (7), 517-529 (2003).</li><li>Miyata, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mitochondrial+free+Ca2++concentration+in+living+single+rat+cardiac+myocytes.">Measurement of mitochondrial free Ca2+ concentration in living single rat cardiac myocytes.</a> American Journal of Physiology. 261 (4 Pt 2), H1123-H1134 (1991).</li><li>Allen, S. P., Stone, D., McCormack, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+loading+of+fura-2+into+mitochondria+in+the+intact+perfused+rat+heart+and+its+use+to+estimate+matrix+Ca2++under+various+conditions.">The loading of fura-2 into mitochondria in the intact perfused rat heart and its use to estimate matrix Ca2+ under various conditions.</a> Journal of Molecular Cellular Cardiology. 24 (7), 765-773 (1992).</li><li>Griffiths, E. J., Halestrap, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrophosphate+metabolism+in+the+perfused+heart+and+isolated+heart+mitochondria+and+its+role+in+regulation+of+mitochondrial+function+by+calcium.">Pyrophosphate metabolism in the perfused heart and isolated heart mitochondria and its role in regulation of mitochondrial function by calcium.</a> Biochemical Journal. 290 (Pt 2), 489-495 (1993).</li><li>Crompton, M., Moser, R., Ludi, H., Carafoli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interrelations+between+the+transport+of+sodium+and+calcium+in+mitochondria+of+various+mammalian+tissues.">The interrelations between the transport of sodium and calcium in mitochondria of various mammalian tissues.</a> European Journal of Biochemistry. 82 (1), 25-31 (1978).</li><li>Chance, B., Schoener, B., Oshino, R., Itshak, F., Nakase, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation-reduction+ratio+studies+of+mitochondria+in+freeze-trapped+samples.+NADH+and+flavoprotein+fluorescence+signals.">Oxidation-reduction ratio studies of mitochondria in freeze-trapped samples. NADH and flavoprotein fluorescence signals.</a> The Journal of Biological Chemistry. 254 (11), 4764-4771 (1979).</li><li>Eng, J., Lynch, R. M., Balaban, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinamide+adenine+dinucleotide+fluorescence+spectroscopy+and+imaging+of+isolated+cardiac+myocytes.">Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes.</a> Biophysical Journal. 55 (4), 621-630 (1989).</li><li>Brandes, R., Bers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+measurements+of+mitochondrial+NADH+and+Ca(2+)+during+increased+work+in+intact+rat+heart+trabeculae.">Simultaneous measurements of mitochondrial NADH and Ca(2+) during increased work in intact rat heart trabeculae.</a> Biophysical Journal. 83 (2), 587-604 (2002).</li><li>Jo, H., Noma, A., Matsuoka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-mediated+coupling+between+mitochondrial+substrate+dehydrogenation+and+cardiac+workload+in+single+guinea-pig+ventricular+myocytes.">Calcium-mediated coupling between mitochondrial substrate dehydrogenation and cardiac workload in single guinea-pig ventricular myocytes.</a> Journal of Molecular Cellular Cardiology. 40 (3), 394-404 (2006).</li><li>Lee, J. H., Ha, J. M., Leem, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Nicotinamide+Adenine+Dinucleotide+Correction+Method+for+Mitochondrial+Ca2++Measurement+with+FURA-2-FF+in+Single+Permeabilized+Ventricular+Myocytes+of+Rat.">A Novel Nicotinamide Adenine Dinucleotide Correction Method for Mitochondrial Ca2+ Measurement with FURA-2-FF in Single Permeabilized Ventricular Myocytes of Rat.</a> Korean Journal of Physiology and Pharmacology. 19 (4), 373-382 (2015).</li><li>Powell, T., Terrar, D. A., Twist, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+properties+of+individual+cells+isolated+from+adult+rat+ventricular+myocardium.">Electrical properties of individual cells isolated from adult rat ventricular myocardium.</a> Journal of Physiology. 302, 131-153 (1980).</li><li>Sun, B., Leem, C. 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The authors have no conflicts of interest to disclose.

The interference correction method was successfully developed for measuring the signals of NADH and fura-2 analogs. Exact measurement of the signals is essential for exact correction. However, the inherent nature of the fluorescent device produces a background signal unrelated to that of NADH of fura-2. The highest quality band-pass filter can only pass up to 10&#8722;8 of the unwanted wavelengths of the light. However, the fluorescent signal from a single cell is very small, and the reflection of the excitati...

The significant role of intracellular Ca2+ is widely known1. The quantification of [Ca2+] is essential to understand the processes of the cellular physiological functions. Fura-2 analogs are quite useful because they are excited in the UV range (&#60;400 nm), and the ratiometric method can be applied for the quantitative measurement. Therefore, other physiological parameters such as pH, membrane potential, etc., can be measured with other fluorescent dyes. The mitochondrial Ca2+ concentration ([Ca2+]m) range was reportedly 0.08&#8722;20 &#956;M2,3,4,5. Among fura-2 analogs, fura-2-FF is appropriate for measuring this range of [Ca2+]. However, the live cells unfortunately contain NADH/NADPH for their metabolic processes, and NADH generates signal interference because of the overlapping excitation and emission spectra with the fura-2 analog. This interference greatly limits the use of fura-2 analogs. Specifically, if the analog is applied to measure mitochondrial [Ca2+], this interference is the biggest obstacle because the highest amount of NADH is in the mitochondria. This is further complicated by NADH changes being related to the mitochondrial membrane potential (&#936;m) and the change of &#936;m affects [Ca2+]m6,7,8,9. Furthermore, for studying [Ca2+]m dynamics, it is essential to know the status of other mitochondrial parameters, such as NADH, &#936;m, and pH.
The emissions at 450 nm and 500 nm with excitations at 353 nm, 361 nm, and 400 nm contain the signals from NADH and fura-2-FF, and the equations are as follows. Herein, 353 nm and 361 nm are the isosbestic points of fura-2-FF for emissions at 450 nm and at 500 nm, respectively.
F361,450 = F361,450,NADH + F361,450,Fura&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;Equation 1 
F353,500 = F353,500,NADH + F353,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 2 
F400,500 = F400,500,NADH + F400,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 3
where Fx,y is the measured emission intensity at y-nm by x-nm excitation, Fx,y,NADH represents the pure NADH-dependent emission intensity, and Fx,y,Fura represents the pure fura-2-FF-dependent emission intensity. Under the same concentration of the fluorescent dye, a certain excitation intensity should produce the same emission intensity. Therefore, the emission intensity ratio of two different excitation wavelengths should be constant. Ca2+ and fura-2 did not affect NADH fluorescence characteristics; therefore, the ratio of the emission at 450 nm and at 500 nm of NADH was constant at any excitation wavelength. The same rule can be used for fura-2-FF based on the assumption that NADH or [Ca2+] does not affect the emission and excitation spectra of fura-2-FF. However, Ca2+ caused a spectral shift of the fura-2-FF emission. Therefore, to remove the effect of Ca2+, isosbestic excitation, which is independent of Ca2+, needs to be used. Each emission wavelength (i.e., 450 nm and 500 nm) has a different isosbestic point, and from our experimental setup, 353 nm at 500 nm and 361 nm at 450 nm were chosen. From these, the following equations are valid10.
Rf = F361,450,Fura/F353,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 4 
RN1 = F400,500,NADH/F361,450,NADH &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 5 
RN2 = F353,500,NADH/F361,450,NADH &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 6
With these constants, the following equations from (Equation 1) (Equation 2), and (Equation 3) are valid.
F361,450 = F361,450,NADH + Rf &#215; F353,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 7 
F353,450 = RN2 &#215; F361,450,NADH + F353,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 8 
F400,500 = RN1 &#215; F361,450,NADH + F400,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 9
From these equations, if Rf, RN1, and RN2 are known, pure signals of NADH and fura-2 can be obtained as follows.
F361,450,NADH = (F361,450 - Rf &#215; F353,500)/(1 &#8722; Rf &#215; RN2) &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 10 
F353,500,Fura = (RN2 &#215; F361,450 &#8722; F353,500)/(Rf &#215; RN2 &#8722; 1) &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 11 
F400,500,Fura = F400,500 &#8722; RN1 &#215; F361,450,NADH &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; Equation 12 
RFura = F353,500,Fura/F400,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 13
The Ca2+-bound form of fura-2-FF was practically non-fluorescent at the 400 nm excitation wavelength. Based on this property, the following new calibration equation can be derived.
[Ca2+] = Kd &#8729; (F400,500,max/F353,500,max) &#215; (RFura &#8722; Rmin) &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 14
where Kd is a dissociation constant, F400,500,max and F353,500,max are the maximum values of the emitted signals at 500 nm with excitations at 400 nm and 353 nm, respectively, and Rmin is the minimum RFura in Ca2+-free condition. Since the isosbestic excitations were used, the equation can be simplified further as follows.
[Ca2+] = Kd &#8729; (1 / Rmin) &#8729; (RFura &#8722; Rmin) &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 15
Therefore, only Kd and Rmin values are required to calculate [Ca2+].

<table>
	<tbody>
		<tr>
			<td>2 mL eppendorf tube</td>
			<td>Axygen</td>
			<td>MCT-200-C</td>
			<td>2 mL Tube</td>
		</tr>
		<tr>
			<td>AD/DA converter</td>
			<td>Instrutech</td>
			<td>ITC-18</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>ADP, Adenosine 5&#8242;-diphosphate monopotassium salt dihydrate</td>
			<td>Sigma-aldrich</td>
			<td>A5285</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Band pass filter</td>
			<td>Ealing Electro-Optics, Inc</td>
			<td>35-3920</td>
			<td>Equipment, 640&#177;11nm</td>
		</tr>
		<tr>
			<td>Band pass filter</td>
			<td>Omega Optical</td>
			<td>690-9823</td>
			<td>Equipment, 590&#177;15nm</td>
		</tr>
		<tr>
			<td>Band pass filter</td>
			<td>Omega Optical</td>
			<td>500DF20-9916</td>
			<td>Equipment, 500&#177;20nm</td>
		</tr>
		<tr>
			<td>Band pass filter</td>
			<td>Chroma Technology Corp.</td>
			<td>60685</td>
			<td>Equipment, 450&#177;30nm</td>
		</tr>
		<tr>
			<td>Calcium chloride solution</td>
			<td>Sigma-aldrich</td>
			<td>21114</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>carboxy-SNARF-1(AM)</td>
			<td>Invitrogen</td>
			<td>C1272</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Charge-coupled device (CCD) camera</td>
			<td>Philips</td>
			<td>FTM1800NH/HGI</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Dichroic mirror</td>
			<td>Chroma Technology Corp.</td>
			<td>86009</td>
			<td>Equipment, Multiband dichroic mirror, Reflection : &#60;400nm, 490&#177;10, 560&#177;10, Transmission : 460&#177;15, 510&#177;20, &#62;580nm</td>
		</tr>
		<tr>
			<td>Dichroic mirror</td>
			<td>Chroma Technology Corp.</td>
			<td>567DCXRU</td>
			<td>Equipment, Reflection : &#60;560nm, Transmission : &#62; 580 nm</td>
		</tr>
		<tr>
			<td>Dichroic mirror</td>
			<td>Chroma Technology Corp.</td>
			<td>480dclp</td>
			<td>Equipment, Reflection : &#60;470nm, Transmission : &#62; 490 nm</td>
		</tr>
		<tr>
			<td>Dichroic mirror</td>
			<td>Chroma Technology Corp.</td>
			<td>20728</td>
			<td>Equipment, Multiband dichroic mirror, Reflection : &#60;405nm, 470&#177;30, Transmission : 430nm~520nm, &#62; 640 nm</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide(DMSO)</td>
			<td>Sigma-aldrich</td>
			<td>154938</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>DMEM, Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td>Sigma-aldrich</td>
			<td>D5030</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>EGTA, Egtazic acid, Ethylene-bis(oxyethylenenitrilo)tetraacetic acid, Glycol ether diamine tetraacetic acid</td>
			<td>Sigma-aldrich</td>
			<td>E4378</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>FCCP, Mesoxalonitrile 4-trifluoromethoxyphenylhydrazone</td>
			<td>Sigma-aldrich</td>
			<td>21857</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>field diaphragm</td>
			<td>Nikon</td>
			<td>86506</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Fura-2-FF(AM)</td>
			<td>TEFLABS</td>
			<td>137</td>
			<td>chemicals</td>
		</tr>
		<tr>
			<td>Green tube</td>
			<td>DWM</td>
			<td></td>
			<td>test tube</td>
		</tr>
		<tr>
			<td>HEPES,&#160; 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N&#8242;-(2-ethanesulfonic acid)</td>
			<td>Sigma-aldrich</td>
			<td>H3375</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>High-speed counter</td>
			<td>National Instruments</td>
			<td>NI-6022</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Hot mirror</td>
			<td>Chroma Technology Corp.</td>
			<td>21002</td>
			<td>Equipment, 50:50</td>
		</tr>
		<tr>
			<td>Inverted microscope </td>
			<td>Nikon</td>
			<td>TE-300</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Malate</td>
			<td>Sigma-aldrich</td>
			<td>27606</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Near infrared filter </td>
			<td>Chroma Technology Corp.</td>
			<td>D750/100X</td>
			<td>Equipment, 750&#177;100nm</td>
		</tr>
		<tr>
			<td>Oil immersion lens </td>
			<td>Nikon</td>
			<td>MRF01400</td>
			<td>40x, NA 1.3; Equipment</td>
		</tr>
		<tr>
			<td>Photon counter unit</td>
			<td>Hamamatsu</td>
			<td>C3866</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Photon multiplier tube</td>
			<td>Hamamatsu</td>
			<td>R2949</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Polychrome II</td>
			<td>Till Photonics</td>
			<td>SA3/MG04</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Merck</td>
			<td>1.04936</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Potassium hydroxide solution</td>
			<td>Sigma-aldrich</td>
			<td>P4494</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>Sigma-aldrich</td>
			<td>107360</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-aldrich</td>
			<td>R8875</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma-aldrich</td>
			<td>S4521</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>TMRE, Tetramethylrhodamine, ethyl ester </td>
			<td>Molecular probes</td>
			<td>T669</td>
			<td>Chemicals</td>
		</tr>
	</tbody>
</table>

a novel nicotinamide adenine dinucleotide correction method for intracellular ca2+ measurement with fura-2-analog in live cells

To measure [Ca2+] quantitatively, fura-2 analogs, which are ratiometric fluoroprobes, are frequently used. However, dye usage is intrinsically limited in live cells because of autofluorescence interference, mainly from nicotinamide adenine dinucleotide (NADH). More specifically, this is a major obstacle when measuring the mitochondrial [Ca2+] quantitatively using fura-2 analogs because the majority of NADH is in the mitochondria. If the fluorescent dye concentration is the same, a certain excitation intensity should produce the same emission intensity. Therefore, the emission intensity ratio of two different excitation wavelengths should be constant. Based on this principle, a novel online correction method of NADH signal interference to measure [Ca2+] was developed, and the real signal intensity of NADH and fura-2 can be obtained. Further, a novel equation to calculate [Ca2+] was developed with isosbestic excitation or excitation at 400 nm. With this method, changes in mitochondrial [Ca2+] could be successfully measured. In addition, with a different set of the excitation and emission wavelengths, multiple parameters, including NADH, [Ca2+], and pH or mitochondrial membrane potential (&#936;m), could be simultaneously measured. Mitochondrial [Ca2+] and &#936;m or pH were measured using fura-2-FF and tetramethylrhodamine ethyl ester (TMRE) or carboxy-seminaphtorhodafluor-1 (carboxy-SNARF-1).

Mitochondrial Ca2+ changes due to correction10 
Figure 4 shows the changes in [Ca2+]m before and after the correction. The results clearly showed the substantial changes in [Ca2+]m. The mitochondrial resting calcium concentration without cytosolic Ca2+ ([Ca2+]c) was 1.03 &#177; 0.13 &#181;M (mean &#177; S.E., n = 32), and the maximum [Ca2+]m at 1-&#181;M [Ca2+</s...

Watch this Scientific Journal Video about A Novel Nicotinamide Adenine Dinucleotide Correction Method for Intracellular Ca2+ Measurement with Fura-2-Analog in Live Cells at JoVE.com

A Novel Nicotinamide Adenine Dinucleotide Correction Method for Intracellular Ca2+ Measurement with Fura-2-Analog in Live Cells

Due to the spectral overlapping of the excitation and emission wavelengths of NADH and fura-2 analogs, the signal interference from both chemicals in live cells is unavoidable during quantitative measurement of [Ca2+]. Thus, a novel online correction method of NADH signal interference to measure [Ca2+] was developed.

A Novel Nicotinamide Adenine Dinucleotide Correction Method for Intracellular Ca2+ Measurement with Fura-2-Analog in Live Cells

To measure [Ca2+] quantitatively, fura-2 analogs, which are ratiometric fluoroprobes, are frequently used. However, dye usage is intrinsically limited in ...

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Research

JoVE Journal

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6.7K Views.  University of Ulsan College of Medicine/Asan Medical Center. Due to the spectral overlapping of the excitation and emission wavelengths of NADH and fura-2 analogs, the signal interference from both chemicals in live cells is unavoidable during quantitative measurement of [Ca2+]. Thus, a novel online correction method of NADH signal interference to measure [Ca2+] was developed.

Video: A Novel Nicotinamide Adenine Dinucleotide Correction Method for Intracellular Ca2+ Measurement with Fura-2-Analog in Live Cells

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

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.

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...