K.-H.K was supported by the Heart and Stroke Foundation of Canada Grant-in-Aid (G-18-0022213), J. P. Bickell Foundation and the University of Ottawa Heart Institute Start-up fund; H.-K.S. was supported by grants from the Canadian Institutes of Health Research (PJT-162083), Reuben and Helene Dennis Scholar and Sun Life Financial New Investigator Award for Diabetes Research from Banting &#38; Best Diabetes Centre (BBDC) and Natural Sciences and Engineering Research Council (NSERC) of Canada (RGPIN-2016-06610). R.Y.K. was supported by a fellowship from the University of Ottawa Cardiology Research Endowment Fund. J.H.L. was supported by the NSERC Doctoral Scholarship and Ontario Graduate Scholarship. Y.O. was supported by UOHI Endowed Graduate Award and Queen Elizabeth II Graduate Scholarship in Science and Technology.

All methods and protocols here have been approved by Animal Care Committees in The Animal Care and Veterinary Service (ACVS) of the University of Ottawa and The Centre for Phenogenomics (TCP) and conform to the standards of the Canadian Council on Animal Care. It should be noted that all procedures described here should be performed under institutional and governmental approval as well as by staff who are technically proficient. All mice were housed in standard vented cages in temperature- and humidity-controlled rooms with 12 h/12 h light/dark cycles (21&#8211;22 &#176;C, 30%&#8211;60% humidity for normal housing) and free access to water. Male C57BL/6J and ob/ob mice were obtained from the Jackson Laboratory.
1. 2:1 Isocaloric IF Regimen
<ol>
	<li>For lean and diet-induced obesity mouse models, prepare either a normal diet (17% fat, ND) or high fat diet (45% fat, HFD). 
	NOTE: 60% HFD can be used to induce severe diet-induced obesity; yet, due to the softness of the food pellet, it is relatively difficult to accurately measure daily food intake. An automated continuous measurement system can improve versatility for multiple types of diets.</li>
	<li>Measure baseline body weight and body composition of each mouse at 7 weeks of age using a scale and EchoMRI, respectively. 
	NOTE: Refer to section 3 for body composition measurement.</li>
	<li>Based on body weight and body composition results, randomly and equally divide 7 week-old male C57BL/6J mice into two groups: ad libitum (AL) and intermittent fasting (IF) groups.</li>
	<li>Place two to three mice per cage and ensure free access to drinking water. 
	NOTE: The number of mice per cage can affect food intake behavior. It is recommended to maintain an equal number of mice per cage in all groups during the study.</li>
	<li>Provide 1 week of acclimation to the new cage environment and diet before starting the IF regimen.</li>
	<li>Fasting period: move mice to a clean cage with fresh bedding at 12:00 PM. Do not add food for the IF group, while providing a weighed amount of food to the AL group. 
	NOTE: For each fasting cycle, it is important to change cages for both AL and IF groups to ensure that both groups are exposed to the same amount of handling time.</li>
	<li>After 24 h, measure the weights of mice in both groups and leftover food in AL cages. 
	NOTE: Make sure to include the weight of food crumbs on the food hopper and bottom of the cage, especially when using HFD, as mice often remove small pellets or fragments of food from the hopper and keep them near nest sites. The average energy intake per mouse at the end of each 2:1 cycle (3 days) is around 35 kcal, equivalent to ~10 g for a normal diet (3.3 kcal/g) and ~7 g for HFD (4.73 kcal/g).</li>
	<li>Feeding period: provide a weighed amount of food at 12:00 PM for both AL and IF groups.</li>
	<li>After 48 h of providing the food, measure the weight of leftover food and mice.</li>
	<li>Repeat steps 1.6&#8211;1.10 for the duration of the study (e.g., 16 weeks).</li>
</ol>
2. Pair-feeding (PF) Control Group
NOTE: For an IF experiment in which altered feeding behavior is observed in a mouse model (e.g., hyperphagia in ob/ob mice), it is necessary to have a pair-feeding group as a control for proper calorie-independent comparison to IF.
<ol>
	<li>For the PF control group, stagger the experiment schedule such that the same amount of food consumed by the IF group is offered to the PF group (Figure 2).</li>
	<li>Measure the amount of food consumed by the IF group over 2 days of refeeding period.</li>
	<li>Divide this amount of consumed food in the IF group evenly into three proportions and provide it daily to the PF group at 12:00 PM. 
	NOTE: Providing an equal amount of food daily is critical. In the case of mice with hyperphagia, if the pair-fed mice are provided with an amount of food less than their voluntary consumption at once, they will likely consume all provided food and become effectively fasted. This may then prevent proper comparison to IF-treated mice and confound the result.</li>
	<li>Repeat steps 2.1&#8211;2.3 for the duration of the study.</li>
</ol>
3. Body Composition Analysis
NOTE: Since long-term IF affects body weight in mice, body composition can be measured at appropriate cycles (e.g., every 3 or 4 cycles) using a body composition analyzer to quantify fat and lean mass in live, non-anesthetized mice.
<ol>
	<li>Turn on the body composition analyzer. 
	NOTE: Before starting the program, leave the machine on for at least 2&#8211;3 h to warm up.</li>
	<li>Run a system test on the body composition analyzer to test its measurement accuracy. If necessary, calibrate the system using canola oil and water samples.</li>
	<li>Measure the body weight of each mouse.</li>
	<li>Place the mouse in a small animal cylindric holder.</li>
	<li>Insert a delimiter to constrain physical movement of the mouse during the measurement and place the holder into the body composition analyzer.</li>
	<li>Run the scanning program. 
	NOTE: It takes approximately 90&#8211;120 s to analyze.</li>
	<li>After measurement, remove the holder from the equipment and bring the mouse back to the cage. 
	NOTE: A more detailed protocol can be found in a previous publication9.</li>
</ol>
4. Glucose and Insulin Tolerance Tests
<ol>
	<li>For glucose tolerance test (GTT), measure body weight and body composition of each mouse before subjecting to fasting and mark the tail with a permanent marker for easy and rapid indexing.</li>
	<li>Place mice in new cages without food at 7:00 PM for overnight fasting. 
	NOTE: Overnight fasting is the standard protocol, yet due to mouse physiology (e.g., increased glucose utilization after prolonged fasting10,11), shorter fasting (~6 h) can be used as described for ITT.</li>
	<li>After fasting 14&#8211;16 h (9:00 AM in the following morning), measure body weight and body composition of each mouse and calculate the amount of glucose dosage based on body weight. 
	NOTE: To avoid overestimation of glucose intolerance in obese mice, lean mass obtained from the body composition analysis can be used to calculate glucose dosage12,13.</li>
	<li>For each mouse, cut the tip of the tail (0.5&#8211;1.0 mm) using clean surgical scissors. After wiping off the first drop of blood, draw a fresh drop of blood from the tail and measure baseline fasting blood glucose level with the glucometer. 
	NOTE: Additional tail cutting is not required for every blood glucose measurement during GTT or ITT. The wound can be freshened by abrading it with gauze to draw a drop of blood.</li>
	<li>Subject mice to an intraperitoneal (i.p.) injection of glucose (1 mg/g of body weight). 
	NOTE: Based on the objective of an experiment (e.g., examining incretin effects), oral administration of glucose can be performed by oral gavage. The protocol for oral GTT (OGTT) can be found in another study14.</li>
	<li>Measure blood glucose from the tail at 0, 5, 15, 30, 60, and 120 min post-glucose injection.</li>
	<li>After finishing the GTT, provide a sufficient amount of food.</li>
	<li>For the insulin tolerance test (ITT), remove food at 9:00 AM. 
	NOTE: Since both GTT and ITT are stress-inducing experiences for mice that can elevate blood glucose levels and change physiology, it is recommended to perform ITT after providing at least 2&#8211;3 days of recovery after GTT experiment.</li>
	<li>After fasting for 6 h (3:00 PM), measure baseline blood glucose from the tail as described in step 4.4.</li>
	<li>Subject mice to i.p. injection of insulin (0.65 mU/g of body weight).</li>
	<li>Measure blood glucose from the tail at 0, 15, 30, 60, 90, and 120 min post-insulin injection.</li>
	<li>After finishing ITT, provide a sufficient amount of food.</li>
</ol>
5. Indirect Calorimetry
NOTE: Energy metabolism of IF-treated mice can be further evaluated through indirect calorimetry over a single cycle of IF. This will measure oxygen consumption (VO2), carbon dioxide production (VCO2), respiratory exchange ratio (RER), and heat (kcal/h).
<ol>
	<li>Turn on the power of the indirect calorimeter system at least 2 h before running the experiment. 
	NOTE: This system warm-up is important for accurate measurement.</li>
	<li>Prepare cages with clean bedding, fill water bottles, and add the pre-weighed amount of chow to the food hoppers.</li>
	<li>Check the condition of the Drierite and lime soda. If a color indicator of the Drierite appears pink, which indicates that the Drierite has absorbed a high amount of moisture, it is necessary to replace or top with fresh Drierite.</li>
	<li>Calibrate the system using a gas with the specific composition (0.5% CO2, 20.5% O2).</li>
	<li>Measure body weight and body composition of each mouse, which will be used to normalize VO2 and VCO2 data.</li>
	<li>Gently place one mouse per cage.</li>
	<li>Assemble metabolic cages, place them in the temperature-controlled environment chamber, and connect to gas lines and activity sensor cable.</li>
	<li>After setting up the experiment profile by adding appropriate experimental parameters using the software, run the program for measurement. The purpose of the first day&#39;s measurement is to provide a period of acclimatization and measure baseline energy metabolism.</li>
	<li>At 12:00 PM the following day, subject mice to 24 h of fasting by removing food and crumbs from the hopper and bottom of the cage. If necessary, replace with clean bedding.</li>
	<li>After 24 h, add the pre-weighed amount of chow to the food hopper for the refeeding period.</li>
	<li>Continue to measure for the next 48 h. Check regularly whether the system is running without hardware or software interruption.</li>
	<li>After completing measurement, terminate the program and bring mice back to their original cages. Measure the amount of leftover food to examine food intake.</li>
	<li>The detailed protocol for indirect calorimetry can be found in a previous study9.</li>
</ol>

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The authors have nothing to disclose.

It has been well-documented that IF provides beneficial health effects on various diseases in both humans and animals8,15,16,17,18,19. Its underlying mechanisms, such as autophagy and gut microbiome, have recently been elucidated. The presented protocol describes an isocaloric 2:1 IF regimen in mice for investigating calorie-...

Modern lifestyle is associated with longer daily food intake time and shorter fasting periods1. This contributes to the current global obesity epidemic, with metabolic disadvantages seen in humans. Fasting has been practiced throughout human history, and its diverse health benefits include prolonged lifespan, reduced oxidative damage, and optimized energy homeostasis2,3. Among several ways to practice fasting, periodic energy deprivation, termed intermittent fasting (IF), is a popular dietary method that is widely practiced by the general population due to its easy and simple regimen. Recent studies in preclinical and clinical models have demonstrated that IF can provide health benefits comparable to prolonged fasting and caloric restriction, suggesting that IF can be a potential therapeutic strategy for obesity and metabolic diseases2,3,4,5.
IF regimens vary in terms of fasting duration and frequency. Alternate day fasting (i.e., 1 day feeding/1 day fasting; 1:1 IF) has been the most commonly used IF regimen in rodents to study its beneficial health impacts on obesity, cardiovascular diseases, neurodegenerative diseases, etc.2,3. However, as shown in previous studies6,7, and further mechanistically confirmed in our energy intake analysis8, 1:1 IF results in underfeeding (~80%) due to the lack of sufficient feeding time to compensate for energy loss. This makes it unclear whether the health benefits conferred by 1:1 IF are mediated by calorie restriction or modification of eating patterns. Therefore, a new IF regimen has been developed and is shown here, comprising of a 2 day feeding/1 day fasting (2:1 IF) pattern, which provides mice with sufficient time to compensate for food intake (~99%) and body weight. These mice are then compared to an ad libitum (AL) group. This regimen enables examination of the effects of isocaloric IF in the absence of caloric reduction in wild-type mice.
In contrast, in a mouse model that exhibits altered feeding behavior, AL feeding may not be a proper control condition to compare and examine the effects of 2:1 IF. For example, since ob/ob mice (a commonly used genetic model for obesity) exhibit hyperphagia due to the lack of leptin regulating appetite and satiety, those with 2:1 IF exhibit ~20% reduced caloric intake compared to ob/ob mice with AL feeding. Thus, to properly examine and compare the effects of IF in ob/ob mice, a pair-feeding group as a suitable control needs to be employed.
Overall, a comprehensive protocol is provided to perform isocaloric 2:1 IF, including use of a pair-feeding control. It is further demonstrated that isocaloric 2:1 IF protects mice from high fat diet-induced obesity and/or metabolic dysfunction in both wild-type and ob/ob mice. This protocol can be used to examine the beneficial health impacts of 2:1 IF on various pathological conditions including neurological disorders, cardiovascular diseases, and cancer.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Comprehensive Lab Animal Monitoring System (CLAMS)</td>
			<td>Columbus Instruments</td>
			<td></td>
			<td>Indirect calorimeter</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose solution</td>
			<td>Sigma-Aldrich</td>
			<td>G8769</td>
			<td>For GTT</td>
		</tr>
		<tr>
			<td>EchoMRI 3-in-1</td>
			<td>EchoMRI</td>
			<td>EchoMRI 3-in-1</td>
			<td>Body composition analysis</td>
		</tr>
		<tr>
			<td>Glucometer and strips</td>
			<td>Bayer</td>
			<td>Contour NEXT</td>
			<td>These are for GTT and ITT experiments</td>
		</tr>
		<tr>
			<td>High Fat Diet (45% Kcal% fat)</td>
			<td>Research Diets Inc.</td>
			<td>#D12451</td>
			<td>3.3 Kcal/g</td>
		</tr>
		<tr>
			<td>High Fat Diet (60% Kcal% fat)</td>
			<td>Research Diets Inc.</td>
			<td>#D12452</td>
			<td>4.73 Kcal/g</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>El Lilly</td>
			<td>Humulin R</td>
			<td>For ITT</td>
		</tr>
		<tr>
			<td>Mouse Strain: B6.Cg-Lepob/J</td>
			<td>The Jackson Laboratory</td>
			<td>#000632</td>
			<td>Ob/Ob mouse</td>
		</tr>
		<tr>
			<td>Mouse Strain: C57BL/6J</td>
			<td>The Jackson Laboratory</td>
			<td>#000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal chow (17% Kcal% fat)</td>
			<td>Harlan</td>
			<td>#2918</td>
			<td></td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Mettler Toledo</td>
			<td></td>
			<td>Body weight and food intake measurement</td>
		</tr>
	</tbody>
</table>

assessment of the metabolic effects of isocaloric 2:1 intermittent fasting in mice

Intermittent fasting (IF), a dietary intervention involving periodic energy restriction, has been considered to provide numerous benefits and counteract metabolic abnormalities. So far, different types of IF models with varying durations of fasting and feeding periods have been documented. However, interpreting the outcomes is challenging, as many of these models involve multifactorial contributions from both time- and calorie-restriction strategies. For example, the alternate day fasting model, often used as a rodent IF regimen, can result in underfeeding, suggesting that health benefits from this intervention are likely mediated via both caloric restriction and fasting-refeeding cycles. Recently, it has been successfully demonstrated that 2:1 IF, comprising 1 day of fasting followed by 2 days of feeding, can provide protection against diet-induced obesity and metabolic improvements without a reduction in overall caloric intake. Presented here is a protocol of this isocaloric 2:1 IF intervention in mice. Also described is a pair-feeding (PF) protocol required to examine a mouse model with altered eating behaviors, such as hyperphagia. Using the 2:1 IF regimen, it is demonstrated that isocaloric IF leads to reduced body weight gain, improved glucose homeostasis, and elevated energy expenditure. Thus, this regimen may be useful to investigate the health impacts of IF on various disease conditions.

Figure 1 shows the feeding analyses after 24 h fasting and the comparison between 1:1 and 2:1 intermittent fasting. A 24 h fasting period resulted in a ~10% reduction in body weight, which was fully recovered after 2 days of refeeding (Figure 1A). A 24 h fasting period induced hyperphagia during the subsequent 2 days of refeeding (Figure 1B). Nevertheless, the comparison of energy intake between 1:1...

Watch this Scientific Journal Video about Assessment of the Metabolic Effects of Isocaloric 2:1 Intermittent Fasting in Mice at JoVE.com

Assessment of the Metabolic Effects of Isocaloric 2:1 Intermittent Fasting in Mice

The current article describes a detailed protocol for isocaloric 2:1 intermittent fasting to protect and treat against obesity and impaired glucose metabolism in wild-type and ob/ob mice.

Intermittent fasting (IF), a dietary intervention involving periodic energy restriction, has been considered to provide numerous benefits and counteract ...

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8.7K Views.  University of Ottawa Heart Institute. The current article describes a detailed protocol for isocaloric 2:1 intermittent fasting to protect and treat against obesity and impaired glucose metabolism in wild-type and ob/ob mice.

Video: Assessment of the Metabolic Effects of Isocaloric 2:1 Intermittent Fasting in Mice

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...