Name: analyzing oxygen consumption rate in primary cultured mouse neonatal cardiomyocytes using an extracellular flux analyzer
Uploaded: 2019-02-13T13:00:00
Description: Watch this Scientific Journal Video about Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer at JoVE.com

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+ ...

<ol>
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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.

<table border="0" cellpadding="0" cellspacing="0" style="width:924px;" width="924">
	<tbody>
		<tr height="28">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
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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 ...

This protocol allows us to isolate and culture high bi-BBT, known only to mouse cardiomyocytes. To evaluate mitochondrial function, oxygen consumption rate of cardiomyocytes can be analyzed by an actual silver flux analyzer. One of the advantages of this technique is that oxygen consumption of cardiomyocytes can be easily measured in their adherent state in 96-well format, which enables testing of multiple conditions with a large number of replicates.This vessel provides important insights into the research area of cardiology. In addition to oxygen consumption, we can also study other metabolic parameters, such as greygrices and peracid epoxidation. Achieving high variability of cardiomyocytes is critical for this experiment.This requires efficient digestion of hearts. Since neo-natal cardiomyocytes are very fragile, gentle handling of cardiomyocytes is also important. In the cell culture hood, aliquot 5ml of HBSS to each well of a 6-well cell culture plate and place it on ice.In addition, aliquot 10ml of HBSS to a 10cm cell culture dish, then, prepare 20 ml of Trypsin Predigestion solution in a 50ml sterile conical tube. Keep all the solutions on ice. Next, extract the heart and transfer it immediately to the sterile cell culture dish containing HBSS.Remove any residual lung tissue and larger vessels. Wash the heart in HBSS using gentle agitation. Subsequently, cut the heart with fine scissors into 8 pieces and transfer the heart tissue with forceps into one well of a 6-well cell culture plate with HBSS.Using a moria spoon, wash the heart tissue by transferring it from well to well in the 6-well plate filled with HBSS. Then transfer the heart tissue into a conical tube containing 20ml of Trypsin and incubate with gentle agitation at 4 degree celsius for 4 hours. In this procedure, prewarm the collagenase digestion solution in a water bath at 37 degrees celsius.Transfer the conical tube containing heart tissue and predigestion solution from 4 degrees celsius to a cell culture hood. Let the heart tissue sink to the bottom of the tube and remove the predigestion solution by using a 10ml serological pipette. Next, add 10ml of HBSS into the tube.Resuspend the heart tissue with HBSS 2 to 3 times to wash out Trypsin using a 10ml serological pipette. Aspirate HBSS and add 10ml of prewarmed collagenase digestion solution into the tube with heart tissue. For the first digestion, incubate the tube with heart tissue in a water bath at 37 degrees celsius for 10 minutes without agitation.Afterward, transfer the tube to the cell culture hood. Triturate the tissue by resuspending it gently 10 times using a 10ml serological pipette. This will allow the heart to disperse and the cells to be released from the heart tissue.Let the undigested tissue sink. Transfer the digested solution enriched in cardiomyocytes to a new conical tube and immediately add an equal amount of cell culture medium to stop the collagenase digestion. Then add 10ml of collagenase digestion solution into the tube containing the remaining undigested heart tissue.For the second digestion, incubate the tube with heart tissue in a 37 degree celsius water bath for 10 minutes. Repeat the procedures of the first digestion and transfer the digested solution enriched in cardiomyocytes to a new conical tube before stopping the collagenase digestion. Next, place a sterile cell strainer in a new sterile 50ml conical tube.Pre-wet the cell strainer with 2 to 3 ml of cell culture medium and pass the cells through the cell strainer. Subsequently, rinse the cell strainer with cell culture medium. Then, centrifuge the conical tube containing cardiomyocytes for 5 minutes at 180 times gravity.After 5 minutes, aspirate the supernatant and resuspend the cell pellet in 10ml of cell culture medium. Plate the cells onto a 10cm cell culture dish and incubate them for one hour. Afterward, gently agitate the plate.Wash off the non-adherent cells and resuspend the cells by repeatedly pipetting the cell culture medium over the dish using a 10ml serological pipette. Then, transfer the non-adherent cells into a new 10cm cell culture dish and incubate them for another hour. After an hour, gently agitate the plate and wash off the non-adherent cells.Transfer the cardiomyocytes into a new 50ml conical tube. Next, prepare a 96-well culture plate by aliquoting 50 microliters of coating solution in each well of the 96-well cell culture plate. If bubbles are present, remove them using a 20 microliter pipette.Incubate the plate at 37 degrees celsius for at least one hour to allow drying of the matrix coating. Then, count the cells using a hemocytometer. Plate the cells into the extra-cellular matrix coated 96-well cell culture plate at a density between 10 to 30 thousand cells per well.And place the cells in the incubater. To preform the oxygen consumption assay, hydrate a flux analyzer sensor cartridge for at least 3 hours, add 200 microliters of calibrant solution into each well of the utility plate. Place the sensor cartridge back onto the utility plate and incubate in 37 degrees celsius without CO2 or O2 supplementation for at least 3 hours.One hour prior to the assay, gently remove the cell culture medium and wash the cells with 200 microliters of prewarmed mitochondrial stress test medium twice. After the second wash, add 175 microliters of prewarmed mitochondrial stress test medium. Then culture the cells at 37 degrees celsius without carbon dioxide or oxygen supplementation.Prepare 3ml of each of the test compounds in mitochondrial stress test medium. Load 25 microliters of each compound into the injector ports of the sensor cartridge using a multi-channel pipette. State of the cells and any pretreatments can affect maximum restoration elicited by injection of uncoupler FCCP.Cell sensitivity to the uncoupler can also change, therefore, it is critical to optimize the working concentration of FCCP for each particular treatment. Subsequently, set up extra-cellular flux assay protocol and start the program. First, put the sensor cartiridge into the machine for calibration.Replace the calibrant for the assay plate once calibration step is done. If desired, after the assay, carefully discard all assay medium using a multi-channel pipette. And store the cell culture mircoplate at negative 20 degrees celsius for future cell normalization using protein assay.By using the protocol described, hearts were isolated from day 0 neo-natal pups and cardiomyocytes were seeded at densities of 10, 20 or 30 thousand cells per well in 96-well plates. After overnight culture, cardiomyocytes were found well attached to the coated plastic surface and there were very few unattached cells. At this point, spontaneously contracting cardiomyocytes were easily visible.A seeding density of 30, 000 cells per well showed confluence one day after seeding as the cells spread. Cardiomyocytes were immunostained with an antibody against sarcomeric alpha actinin, a cardiomyocyte specific marker. As shown here, most of the cells showed positive staining of alpha actinin indicating the high purity of the cardiomyocyte isolation.A scheme of one typical mitochondrial stress test is shown here. The mitochondrial stress test starts with a base line measurement of the oxygen consumption rate, this is followed by the injection of Oligo myosin which inhibits ATPA's. Then, the uncoupling agent FCCP was injected to measure maximum oxygen consumption rate.Finally, with the injection of two electron transport complex inhibitors, mitochondrial respiration completely stops and OCR decreases to its lowest level. To obtain consistent and reproducible results. It is critical to achieve high survivablity.Therefore, it is important to use newborn neonates and gently handled heart tissues in cardiomyocytes. By using new or existing genetically manipulated mouse lines, this method can be easily translated to data that may lead to understanding new mechanisms for bioenergetic regulation in the heart. Mitochondrial play key roles in heart function.We expect this method to help identifying novel regulators and pathways regulating oxidating metabolism and finding new therapeutic targets for treating heart failure.

analyzing-oxygen-consumption-rate-primary-cultured-mouse-neonatal

Research

JoVE Journal

Medicine

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.