We would like to thank all Ross lab and Murphy lab members. This work is supported by American Heart Association (14SDG17790005) to Y.C. NIH (HL115933, HL127806) and VA Merit (BX003260) to R.S.R.

For work with neonatal mice, please refer to local university/institute guidelines set forth by the animal care programs and adhere to one&#8217;s institutional and other appropriate regulations. All methods described in this protocol have been approved by the UC San Diego Institutional Animal Care and Use Committee (IACUC) and adhere to federal and state regulations.
1. Preparation of Reagents
<ol>
	<li>Prepare 25 mL of pre-digestion solution: HBSS (without Ca2+ and Mg2+) supplemented with trypsin (0.5 mg/mL). Sterilize the solution using a 0.22 &#181;m filter and keep on ice until use. Make pre-digestion solution on the day of experiment. 
	NOTE: It is critical to use HBSS without Ca2+ and Mg2+, as Ca2+ and Mg2+ will cause myocyte contraction and subsequent cell death during isolation.</li>
	<li>Prepare 30 mL of collagenase digestion buffer: collagenase (0.8 mg/mL, approximately 350 U/mL) dissolved in HBSS (without Ca2+ and Mg2+) buffer. Sterilize the solution using a 0.22 &#181;m filter and keep on ice until use. Make collagenase digestion solution on the day of experiment.</li>
	<li>Prepare 500 mL of cardiomyocyte culture media (growth media): Mix 375 mL of DMEM, 125 mL of M-199, 25 mL of Horse serum, and 12.5 mL of FBS. Supplement with 1% penicillin and 1% streptomycin solution.</li>
	<li>Prepare mitochondrial stress test medium: Make 200 mL of DMEM based stress test medium (DMEM without NaHCO3, see Table of Materials) supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, and 10 mM glucose, and 2 mM Hepes. 
	NOTE: Make 1 L of medium with DMEM without sodium pyruvate, L-glutamine, glucose, and Hepes, filer sterile, and store in 4 &#176;C. Prepare the stress test medium on the day of the assay by adding other reagents. Using DMEM medium without NaHCO3 is critical.</li>
	<li>Adjust pH of mitochondrial stress test media to 7.4 on the day of use. Warm media to 37 &#176;C before use.</li>
	<li>Prepare oligomycin: Prepare 5 mL of a 5 mM stock solution in DMSO, make 250 &#181;L aliquots, and store at -20 &#176;C.</li>
	<li>Prepare FCCP: Prepare 5 mL of a 5 mM stock solution in DMSO, make 250 &#181;L aliquots, and store at -20 &#176;C.</li>
	<li>Prepare antimycin A: Prepare 5 mL of a 5 mM stock solution in DMSO, make 250 &#181;L aliquots, and store at -20 &#176;C.</li>
	<li>Prepare rotenone: Prepare 5 mL of a 5 mM stock solution in DMSO, make 250 &#181;L aliquots, and store at -20 &#176;C. 
	NOTE: All reagents and solutions used in this protocol are listed in Table 1.</li>
</ol>
2. Harvesting and Pre-digestion of Hearts from Neonatal Mice (Day 1)
<ol>
	<li>Autoclave scissors, forceps, and a Moria spoon to sterilize.</li>
	<li>Perform all steps in the cell culture hood for sterility.</li>
	<li>Aliquot 5 mL of HBSS (without Ca2+, Mg2+) to each well of a 6-well cell culture plate; place on ice. Aliquot 10 mL of HBSS into a 10 cm cell culture dish.</li>
	<li>Prepare 20 mL of trypsin pre-digestion solution in a 50 mL sterile conical tube. Keep all solutions on ice.</li>
	<li>Quickly dip newborn (day 0) mice in 70% ethanol solution for sterilization.</li>
	<li>Decapitate pups using sterile scissors (straight) without anesthesia, and then open chest along the sternum to allow access to the chest cavity and the heart. (Figure 1A) 
	NOTE: 1) It is critical to use P0 neonatal mice to achieve high cell viability. 2) This euthanasia method is permitted for neonates in accordance with NIH and American Veterinary Medical Association guidelines 17.</li>
	<li>Extract hearts from the body with a fine scissors and transfer immediately into the sterile cell culture dish containing HBSS (without Ca2+, Mg2+) (Figure 1B).</li>
	<li>Remove any residual lung tissue, larger vessels, etc. (and atria, if desired). Wash hearts in the HBSS solution using gentle agitation.</li>
	<li>Cut each heart with a fine scissors into 8 pieces and transfer the all heart tissue with forceps into one well of a 6-well cell culture plate with HBSS (Figure 1C and 1D).</li>
	<li>Wash the hearts by transferring the hearts from well to well in the 6-well plate filled with HBSS, using a Moria spoon (Figure 1D). 
	NOTE: Transferring the hearts from well to well is enough to wash out the blood. Since blood interferes with enzymatic digestion it is important to wash the hearts with HBSS and remove blood.</li>
	<li>Transfer the hearts with a Moria spoon into a conical tube containing 20 mL of trypsin (0.5 mg/mL) and incubate with gentle agitation at 4&#176;C for 4 h (Figure 1E).</li>
</ol>
3. Prepare a 96-well Culture Plate (Day 1)
<ol>
	<li>Prepare 5 mL of coating solution: PBS containing 0.5% gelatin (autoclave before use) and 1% fibronectin solution. (e.g. 5 mL gelatin solution plus 50 &#181;L of fibronectin solution)</li>
	<li>Aliquot 50 &#181;L of coating solution into each well of the 96-well cell culture plate (see Table of Materials). If bubbles are present, remove them by using a 20 &#181;L pipette to suck bubbles out. 
	NOTE: It is important to cover all the surface area of each well with coating solution.</li>
	<li>Incubate the plate in a 37 &#176;C cell culture incubator for 1 h or more to allow drying of the matrix coating.</li>
	<li>Aspirate any residual coating solution before seeding cardiomyocytes.</li>
</ol>
4. Enzymatic Digestion and Plating of Cells (Day 1)
<ol>
	<li>Pre-warm collagenase digestion solution in a 37 &#176;C water bath. 
	NOTE: This is step is important to achieve efficient enzymatic digestion.</li>
	<li>Move the conical tube containing hearts and pre-digestion solution from 4 &#176;C to a cell culture hood. (Figure 1F)</li>
	<li>Let the hearts sink to the bottom of the tube and remove the pre-digestion solution by using a 10 mL serological pipette (1 to 2 mL of the isolation medium may remain in the tube).</li>
	<li>Add 10 mL of HBSS into the tube. Re-suspend the hearts with HBSS 2-3 times to wash out trypsin using a 10 mL serological pipette. Aspirate HBSS (1 to 2 mL may remain in the tube).</li>
	<li>Add 10 mL of pre-warmed collagenase digestion solution into the tube with hearts. (Figure 1G)</li>
	<li>Incubate the tube with hearts in a 37 &#176;C water bath for 10 min without agitation (1st digestion).</li>
	<li>After 1st digestion, move the tube to the cell culture hood. Gently triturate hearts by re-suspending the hearts within the tube gently 10 times using a 10 mL serological pipette. This will allow hearts to disperse and cells to be released from heart tissue. (Figure 1H) 
	NOTE: Since cardiomyocytes are fragile, gentle trituration is important to achieve high viability.</li>
	<li>Let the undigested tissue sink, transfer digested solution enriched in cardiomyocytes (approximately 9 - 10 mL) to a new conical tube, and immediately add an equal amount of cell culture media to stop the collagenase digestion.</li>
	<li>Add 10 mL of collagenase digestion solution into the tube containing the remaining undigested heart tissue.</li>
	<li>Incubate the tube with heart tissue in a 37 &#176;C water bath for 10 min (2nd digestion).</li>
	<li>Repeat procedure 4.7 and 4.8. 
	NOTE: If there is still much undigested tissue, repeat digestion one more time. However, in most cases, two digestions are enough to disperse most of the cells from the heart tissue.</li>
	<li>Place a sterile cell-strainer (100 &#181;m nylon mesh) in a new sterile 50 mL conical tube. Pre-wet the cell strainer with 2-3 mL of cell culture media and pass cells through the cell-strainer. Rinse the cell-strainer with 2-3 mL of cell culture media. (Figure 1I)</li>
	<li>Centrifuge conical tube containing cardiomyocytes for 5 min at 180 x g (Figure 1J). Aspirate the supernatant (Figure 1K), which will contain cell tissue debris and re-suspend the cell pellet in 10 mL of cell culture media (Figure 1L).</li>
	<li>Gently resuspend the cells and plate cells onto a 10 cm cell culture dish (plastic without any type of coating) and incubate for 1 h in a cell culture incubator (1st pre-plating) (Figure 1M). This pre-plating step allows non-cardiomyocytes, such as fibroblasts and endothelial cells, to adhere to the uncoated cell-culture dish. 
	NOTE: At this point, cardiomyocytes are typically a round shape and appear shiny under the microscope. (Figure 1N).</li>
	<li>After the 1 h incubation, gently agitate the plate, wash non-adherent cells (enriched in cardiomyocytes) from the 10 cm culture dish, and re-suspend cells by repeatedly pipetting the cell culture medium over the dish using a 10 mL serological pipette. Then, transfer non-adherent cells (enriched in cardiomyocytes) into a new 10 cm cell culture dish (plastic without any coating) and incubate for an additional 1 h in a cell culture incubator (2nd pre-plating). 
	NOTE: Cells that attach to the non-coated plate are dominantly non-cardiomyocytes: fibroblasts and endothelial cells, that can be visualized under a microscope (Figure 1O).</li>
	<li>After 2nd pre-plating, gently agitate the plate, wash non-adherent cells (cardiomyocytes) from the 10 cm culture dish, then transfer the cardiomyocytes into a new 50 mL conical tube.</li>
</ol>
5. Counting Cells and Plating Cells into a 96-well Cell Culture Plate (Day 1)
<ol>
	<li>Count the cells using a hemocytometer.</li>
	<li>Plate the cells into an extracellular matrix coated 96-well cell culture plate at a density between&#160;10&#8211;30 x 103&#160;cells/well by using a multi-channel pipette in a final volume of 200 &#181;L (Figure 1P). Use wells A1, A12, H1, and H12 for background: add 200 &#181;L of culture medium as other wells in these wells (no cells). Incubate the plate in a 37 &#176;C cell culture incubator. 
	NOTE: In this study, oxygen consumption was tested by using different cell densities such as 10 x 103, 20 x 103, or 30&#160;x 103 cells/well. (Figure 2A). Also, as above, cardiomyocytes immediately after isolation are typically a round shape and appear shiny under the microscope. Viable cells will flatten out within 16-24 h of culture.</li>
</ol>
6. Oxygen Consumption Assay using a 96-well-format Extracellular Flux Analyzer (Day 2)
NOTE: Oxygen consumption assay can be carried out one day after plating the cells or later. Neonatal cardiomyocytes cultured using this protocol can survive up to 7 days post isolation.
<ol>
	<li>Hydrate a flux analyzer sensor cartridge (see Table of Materials) for at least 3 hours, but ideally for a full day, before the assay. Add 200 &#181;L of Calibrant solution (see Table of Materials) into the each well of the utility plate, put the sensor cartridge back onto the utility plate, and incubate in a 37&#176;C incubator without CO2 or O2 supplementation.</li>
	<li>Change cell culture media to mitochondria stress test medium one hour prior to the assay. Cardiomyocytes are fragile. Therefore, gently remove the cell culture media by using a multi-channel pipette and wash the cells with 200 &#181;L of pre-warmed mitochondrial stress test media twice. After second wash, add 175 &#181;L of pre-warmed mitochondria stress test media and culture the cells in a 37&#176;C incubator without CO2 or O2 supplementation.</li>
	<li>Prepare concentrated test compounds. For mitochondria stress test, prepare 3.0 mL each of 16 &#181;M oligomycin, 9 &#181;M FCCP, and a mixture of 20 &#181;M rotenone and 20 &#181;M antimycin A, all in mitochondrial stress test medium. 
	NOTE: Each compound at the concentration described has been tested. However, titrating the concentration of each compound in one&#8217;s own laboratory is necessary.</li>
	<li>Load 25 &#181;L of each compound into the injector ports of the sensor cartridge using a multichannel pipette (Figure 1Q). The volume and final concentration are described in Table 2.</li>
	<li>Set up extracellular flux assay protocol. The program is described in Table 3.</li>
	<li>Start the program. First, put the sensor cartridge into the machine for calibration (Figure 1R). Replace the calibrant for the assay plate once the calibration step is done. 
	NOTE: Using the software provided by manufacturer, indicate groups of wells and each compound and port.</li>
	<li>If desired, after the assay, carefully discard all assay medium by using a multi-channel pipette and store the cell culture microplate at -20 &#176;C for future cell normalization using protein assay.</li>
	<li>Measure protein content. Add 50 &#181;L of standard RIPA cell lysis solution (see Table of Materials). Incubate the plate on ice for 30 min to fully lyse cells. Transfer all material to a new clear flat bottom 96 well assay plate.</li>
	<li>Measure protein concentration by BCA assay according to manufacturer&#8217;s protocol. 
	NOTE: Coating the well with extracellular matrix results in a high protein concentration in each well. Therefore, subtract the amount of the background well(s) (no cells) to get actual cell protein concentration. The protein concentration derived from cells is low in a 96-well culture plate. In addition, protein concentration can vary from well-to-well due to extracellular matrix coating. Therefore, using cell number to normalize the OCR is suggested.</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Gene+Expression+Analysis+of+Mouse+and+Human+Cardiac+Maturation.">Comparative Gene Expression Analysis of Mouse and Human Cardiac Maturation.</a> Genomics, Proteomics &amp; Bioinformatics. 14 (4), 207-215 (2016).</li></ol>

The authors declare that they have no competing financial interests.

In this study, we have established a simple protocol for isolating and culturing mouse neonatal cardiomyocytes. By using these cardiomyocytes, we also optimized the conditions to measure oxygen consumption rate by using an extracellular flux analyzer system. The protocol allows one to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the principal working cells of the heart, akin to what would be measured in the intact organ. Our protocol is different from ...

To sustain a continuous cardiac contractile function, cardiomyocytes must maintain a constant supply of cellular energy primarily in the form of ATP1. In the heart, approximately 95% of ATP is generated by mitochondria, mainly through oxidative phosphorylation, showing that mitochondria play a crucial bioenergetic role in cardiac function2,3. Supporting this notion is that dysregulation of mitochondrial function can lead to cardiomyopathy and heart failure4,5. Conversely, restoring mitochondrial function has been shown to improve cardiac function of the failing heart6,7. Therefore, studying the mechanism of mitochondrial bioenergetics and identifying novel regulators of mitochondrial function in cardiomyocytes will not only reveal mechanistic insights of cardiac energy production but also could provide insight that will lead to development of new therapeutic targets to treat heart diseases6,8.
Compared to the whole heart, which contains a mixture of myocytes and non-myocytes9, cardiomyocyte cultures are extremely pure, with minimal contamination of non-myocytes from the heart, such as fibroblasts and endothelial cells10. In addition, isolating cardiomyocytes from neonatal pups enables culturing a large number of cells in a small amount of time, compared to isolating cells from adult hearts10,11. Most importantly, primary cultured adult mouse cardiomyocytes have short survival times (e.g. 24 h) and at longer time points de-differentiate. Neonatal mouse cardiomyocytes can survive and be manipulated for upwards of 7 days in culture, making them ideal for testing the effects of drug compounds and gene manipulation on the function of mitochondria in cardiomyocytes10. Of course, there are significant biological differences between the adult and neonatal cells, but the longer duration available for culture of neonatal cells makes them appropriate for many different types of studies, including those of mitochondrial function.
To date, primary cultured neonatal mouse and rat cardiomyocytes have been used as models to study cardiac bioenergetics12,13. In recent years, studies used an extracellular flux analyzer to measure oxygen consumption rate (OCR) and evaluate oxidative capacity in mouse and rat neonatal cardiomyocytes14,15. While compared to rats, the cell viability of mouse neonatal cardiomyocytes is lower and has greater variability16. Also, the ability to study cells from genetically engineered mouse models makes the mouse cell model very important. Given that OCR studies are so sensitive to cell number and seeding density, development of a reproducible, reliable, and simple protocol to achieve consistent cell yield and viability is needed.
Here, we report an optimized protocol that has been developed which uses cultured mouse neonatal cardiomyocytes along with a 96-well-format extracellular flux analyzer for OCR analysis. This protocol greatly increases reproducibility of the assay. In addition, the protocol not only provides a novel and reproducible method for OCR analysis, but also could be adapted to a larger size culture for other experimental purposes, such as that which may be needed to study myofibrillar functions and intracellular signaling pathways.
In particular, this protocol describes a one-day procedure for isolation and culture of neonatal mouse cardiomyocytes in a 96-well cell culture plate. In addition, it describes the procedure to measure oxygen consumption using an extracellular flux analyzer. All solutions used are sterile or sterile filtered. All tools are sterilized by 75% ethanol. We provide a&#160;Table of Materials for various parts of the procedure. For culturing cardiomyocytes, all procedures and steps are performed in a standard cell culture hood. This protocol is developed for the isolation of neonatal mouse hearts from one litter (approximately 8-10 pups). However, the protocol can also be adapted for isolating cardiomyocytes from multiple litters.

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			<td height="28" style="height:28px;width:269px;">Antimycin A</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">A8674</td>
			<td style="width:288px;">Inhibits complex III of the mitochondria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Cell strainer 100 &#956;m pores</td>
			<td style="width:187px;">FALCON</td>
			<td style="width:180px;">352360</td>
			<td style="width:288px;">To capture undigested tissue</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Collagenase type II</td>
			<td style="width:187px;">Worthington</td>
			<td style="width:180px;">LS004176</td>
			<td style="width:288px;">To make collagenase digestion solution</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:269px;">D-Glucose</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">75351</td>
			<td style="width:288px;">To make mitochodnrial stress test medium</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:269px;">DMEM high glucose</td>
			<td style="width:187px;">Life technologies</td>
			<td style="width:180px;">11965-092</td>
			<td style="width:288px;">To make cell culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">DMEM without NaHCO3, Glucose, pyruvate, glumanine, and Hepes</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">D5030-10X1L</td>
			<td style="width:288px;">To make mitochodnrial stress test medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">FCCP (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone)</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">C2920</td>
			<td style="width:288px;">Uncouples mitochondrial respiration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fetal bovine serum (FBS)</td>
			<td style="width:187px;">Life technologies</td>
			<td style="width:180px;">26140-079</td>
			<td style="width:288px;">To make cell culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Fibronectin from bovine plasma</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">F1141-5MG</td>
			<td style="width:288px;">To make coating solution for tissue culture plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fine scissors</td>
			<td style="width:187px;">Fine Sciences Tools</td>
			<td style="width:180px;">14060-10</td>
			<td style="width:288px;">For dissection of hearts</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Gelatin from porcine skin</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">G-1890</td>
			<td style="width:288px;">To make coating solution for tissue culture plates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">HBSS (Hank&#39;s balnced salt solution,without Ca2+, Mg2+)</td>
			<td style="width:187px;">Cellgro</td>
			<td style="width:180px;">21-022-CV</td>
			<td style="width:288px;">To wash hearts and make pre-digestion and collagnase digestion solution</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:269px;">HEPES (1M)</td>
			<td style="width:187px;">Fisher scientific</td>
			<td style="width:180px;">15630080</td>
			<td style="width:288px;">To make mitochodnrial stress test medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Horse serum</td>
			<td style="width:187px;">Life technologies</td>
			<td style="width:180px;">26050-088</td>
			<td style="width:288px;">To make cell culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">L-Glutamine</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">G-3126</td>
			<td style="width:288px;">To make mitochodnrial stress test medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">M-199</td>
			<td style="width:187px;">Cellgro</td>
			<td style="width:180px;">10-060-CV</td>
			<td style="width:288px;">To make cell culture medium</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:269px;">Moria spoon</td>
			<td style="width:187px;">Fisher scientific</td>
			<td style="width:180px;">NC9190356</td>
			<td style="width:288px;">To wash hearts&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Oligomycin</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">75351-5MG</td>
			<td style="width:288px;">Inhibits mitochondrial ATP synthase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">RIPA buffer</td>
			<td style="width:187px;">Fisher scientific</td>
			<td style="width:180px;">89900</td>
			<td style="width:288px;">To lyse the cells for protein assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Rotenone</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">R8875</td>
			<td style="width:288px;">Inhibits complex I of the mitochondria</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Seahorse XFe96 Extracellular Flux 
			Analyzer</td>
			<td style="width:187px;">Agilent</td>
			<td style="width:180px;"></td>
			<td style="width:288px;">Device used to analyze oxygen consumption rate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Seahorse XFe96 FluxPak</td>
			<td style="width:187px;">Agilent</td>
			<td style="width:180px;">102601-100</td>
			<td style="width:288px;">Package of flux analyzer culture plates, sensor cartridges, and calibrant</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Sodium pyruvate</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">P2256</td>
			<td style="width:288px;">To make mitochodnrial stress test medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Straight scissors</td>
			<td style="width:187px;">Fine Sciences Tools</td>
			<td style="width:180px;">91401-12</td>
			<td style="width:288px;">For dissection of hearts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Syringe filter 0.2 &#956;m size</td>
			<td style="width:187px;"></td>
			<td style="width:180px;"></td>
			<td style="width:288px;">For sterile filtration of digestion medium</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Trypsin</td>
			<td style="width:187px;">USB Corporation</td>
			<td style="width:180px;">22715 25GM</td>
			<td style="width:288px;">To make pre-digestion solution</td>
		</tr>
	</tbody>
</table>

analyzing oxygen consumption rate in primary cultured mouse neonatal cardiomyocytes using an extracellular flux analyzer

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

By using the protocol described, hearts were isolated from day 0 neonatal pups. 5 x 105 cells/pup were obtained, and cardiomyocytes were seeded at densities of 10 x 103, 20 x 103, or 30&#160;x 103 cells/well, in 96 well plates (Figure 2A). After overnight culture, cardiomyocytes were found well-attached to the coated plastic surface and there were very few unattached cells (the unattached cells will still appear as ...

Watch this Scientific Journal Video about Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer at JoVE.com

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...

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8.6K Views.  University of California San Diego. The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

Video: Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of inner ear gene therapy is to find a delivery method which results in consistent transduction efficiency of targeted cell types while minimizing hearing loss. In this study, we describe the posterior semicircular canal approach as a viable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear. The easy anatomic identification of the posterior semicircular canal, as well as minimal manipulation of the temporal bone required, make this surgical approach an attractive option for inner ear gene delivery.

In this study, we describe the posterior semicircular canal approach as a reliable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear.

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of ...

Autonomic Function Following Concussion in Youth Athletes: An Exploration of Heart Rate Variability Using 24-hour Recording Methodology

Participation in organized sports makes a significant contribution to youth development, but places youth at a higher risk for sustaining a concussion. To date, return-to-activity decision-making has been anchored in the monitoring of self-reported concussion symptoms and neurocognitive testing. However, multi-modal assessments that corroborate objective physiological measures with traditional subjective symptom reporting are needed and can be valuable. Heart rate variability (HRV) is a non-invasive physiological indicator of the autonomic nervous system, capturing the reciprocal interplay between the sympathetic and parasympathetic nervous systems. There is a dearth of literature exploring the effect of concussion on HRV in youth athletes, and developmental differences preclude the application of adult findings to a pediatric population. Further, the current state of HRV methodology has primarily included short-term (5-15 min) recordings, by using resting state or short-term physical exertion testing to elucidate changes following concussion. The novelty in utilizing a 24 h&#160;recording methodology is that it has the potential to capture natural variation in autonomic function, directly related to the activities a youth athlete performs on a regular basis. Within a prospective, longitudinal research setting, this novel approach to quantifying autonomic function can provide important information regarding the recovery trajectory, alongside traditional self-report symptom measures. Our objectives regarding a 24 h recording methodology were to (1) evaluate the physiological effects of a concussion in youth athletes, and (2) describe the trajectory of physiological change, while considering the resolution of self-reported post-concussion symptoms. To achieve these objectives, non-invasive sensor technology was implemented. The raw beat-to-beat time intervals captured can be transformed to derive time domain and frequency domain measures, which reflect an individual&#39;s ability to adapt and be flexible to their ever-changing environment. By using non-invasive heart rate technology, autonomic function can be quantified outside of a traditional controlled research setting.

We demonstrate a 24 h heart rate recording methodology to evaluate the influence of concussion across the recovery trajectory in youth athletes, within an ecologically valid context.

Participation in organized sports makes a significant contribution to youth development, but places youth at a higher risk for sustaining a concussion. To ...

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular respiration, oxidative metabolism, signal transduction, and apoptosis. Consequently, mitochondrial dysfunction is reported to play an important role in the antifungal drug tolerance and virulence of pathogenic fungi. Recent data have also led to the recognition of the importance of the mitochondria as an important contributor to fungal pathogenesis. Despite the importance of the mitochondria in fungal biology, standardized methods to understand its function are poorly developed. Here, we present a procedure to study the basal oxygen consumption rate (OCR), a measure of mitochondrial respiration, and extracellular acidification rates (ECAR), a measure of glycolytic function in C. albicans strains. The method described herein can be applied to any Candidaspp. strains without the need to purify mitochondria from the intact fungal cells. Furthermore, this protocol can also be customized to screen for inhibitors of mitochondrial function in C. albicans strains.

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts

Bone development and homeostasis is dependent upon the differentiation and activity of bone forming osteoblasts. Osteoblast differentiation is sequentially characterized by proliferation followed by protein synthesis and ultimately bone matrix secretion. Proliferation and protein synthesis require a constant supply of amino acids. Despite this, very little is known about amino acid consumption in osteoblasts. Here we describe a very sensitive protocol that is designed to measure amino acid consumption using radiolabeled amino acids. This method is optimized to quantify changes in amino acid uptake that are associated with osteoblast proliferation or differentiation, drug or growth factor treatments, or various genetic manipulations. Importantly, this method can be used interchangeably to quantify amino acid consumption in cultured cell lines or primary cells in vitro or in isolated bone shafts ex vivo. Finally, our method can be easily adapted to measure the transport of any of the amino acids as well as glucose and other radiolabeled nutrients.

This protocol presents a radiolabeled amino acid uptake assay, which is useful for evaluating amino acid consumption either in primary cells or in isolated bones.