We thank the Northwestern University Mouse Histology and Phenotyping Laboratoryfor technical support and assistance, and Noboru Mizushima (University of Tokyo) for providing GFP-LC3 transgenic mice. A. R. and C. H. were supported by the startup funds from Northwestern University and the grant from National Institutes of Health (DK094980).

All procedures involving animals were performed according to guidelines approved by the Northwestern University Institutional Animal Care and Use Committee (IACUC).
1. Mouse Models
<ol>
	<li>Use 8-12 week-old mice in the exercise training. To detect exercised-induced autophagy in vivo, use the GFP-LC3 transgenic mice (C57BL/6 background) for imaging studies and C57BL/6 mice for biochemical analyses.</li>
</ol>
2. Exercise-induced Autophagy
<ol>
	<li>Treadmill setup - forced exercise
	<ol>
		<li>Acclimate and train the mice on a 10&#176; uphill open treadmill for 2 days. On day 1, exercise the mice for 5 min at 8 m/min, and on day 2, exercise the mice for 5 min at 8 m/min followed by another 5 min at 10 m/min10. Encourage the mice to run by gentle hand nudge.</li>
		<li>On the 3rd day, let the mice undergo a single bout of running for 90 min on the 10&#176; uphill treadmill, starting at the speed of 10 m/min. After 40 min, increase the treadmill speed at a rate of 1 m/min every 10 min for a total of 30 min, and then increase the speed at the rate of 1 m/min every 5 min until it reaches 17 m/min, for a total of 90 min of exercise time and 1,070 meters of running distance.</li>
		<li>Set the electric stimulus at a low intensity range (1.2 Hz), and encourage the mice to run to avoid repeated foot shock.</li>
		<li>For tissue collection, euthanize the mice by isoflurane inhalation, followed by cervical dislocation immediately after exercise.&#160;Other approved euthanasia methods can also be used, such as CO2 or I.P. injection of other anesthetics. The level of autophagy does not seem to be affected by methods of euthanasia.</li>
	</ol>
	</li>
	<li>Running wheel setup - voluntary exercise
	<ol>
		<li>Place a mouse-running wheel (11.4 cm diameter) inside a cage with a single-housed mouse.</li>
		<li>Verify the running capacity using a bike odometer. Set up the wheel parameter [distance (in mm) per full rotation of the wheel] at 358 (11.4*&#960;). Measure the running distance after 24 hr.</li>
		<li>Let the mouse run voluntarily for 2 weeks with the running wheel. For tissue collection, sacrifice the mouse in the morning after the running period.</li>
	</ol>
	</li>
</ol>
3. Autophagy Flux Evaluation
<ol>
	<li>To measure the autophagic flux under resting and exercise conditions, treat mice with the autophagy inhibitor chloroquine for three days and compare them to mice treated with PBS.
	<ol>
		<li>Dissolve chloroquine in PBS (5 mg/ml), and inject chloroquine into mice intraperitoneally at the dose of 50 mg/kg/day for three consecutive days. Sacrifice mice and collect tissues 3 hr after the last injection.</li>
		<li>For exercised mice, pretreat them with chloroquine for two consecutive days at the dose of 50 mg/kg/day. On the third day, inject the mice with the same concentration of chloroquine, and after 90 min let them run on the treadmill for another 90 min.</li>
		<li>Euthanize the mice by isoflurane inhalation, followed by cervical dislocation. Collect tissues immediately after running, so that the total incubation time of chloroquine is the same as control (resting) mice (3 hr). 
		NOTE: Alternatively, use a single injection of chloroquine to determine the autophagy flux in mouse tissues.</li>
	</ol>
	</li>
</ol>
4. Tissue Harvest and Fixation
<ol>
	<li>Prior to tissue harvest prepare phosphate buffered saline (PBS) and fresh 4% paraformaldehyde (PFA, caution: personal protection equipment required) in PBS at a final pH of 7.4. Store both solutions at 4 &#176;C.</li>
	<li>Fill 30 ml syringe with 15 - 20 ml of 4% PFA (for GFP-LC3 mice). Connect the syringe with a 20 G catheter needle.</li>
	<li>After the exercise, euthanize the mice by isoflurane inhalation, followed by cervical dislocation.</li>
	<li>Place the animal on a clean work surface on its back. Cut and open the thoracic cavity by cutting the diaphragm to expose the heart.</li>
	<li>Make a small incision on the liver to let the blood come out, and inject a total of 15 ml 4% PFA slowly into the right ventricle of the heart via the catheter, until the lung and liver turn completely pale. Perfuse the mouse immediately after the exercise session, using a syringe pump with a constant flow rate of 90 ml/hr.</li>
	<li>After perfusion, remove the needle and collect tissues of interest (e.g. brain11 and skeletal muscle). For skeletal muscle, dissect the vastus lateralis. Briefly, pull the skin of the leg backwards to expose the muscles, localize the quadriceps femoris12, and dissect out vastus lateralis, which is the external muscle attached to the upper portion of the femoral bone.</li>
	<li>For further fixation and dehydration, place the tissues of GFP-LC3 mice in 4% PFA for 24 hr at 4 &#176;C, and then transfer them to 15% sucrose-PBS at 4 &#176;C overnight or until the tissues have settled, and then to 30% sucrose-PBS at 4 &#176;C overnight or for longer storage. Keep the samples in the dark as much as possible as they may be sensitive to strong light.</li>
	<li>For western blot analysis snap freeze the tissues from C57BL/6 mice directly in liquid nitrogen, and store them at -80 &#176;C.</li>
</ol>
5. Imaging Analysis of GFP-LC3 Puncta
<ol>
	<li>Tissue process
	<ol>
		<li>Pre-treat the tissues (previously fixed in PFA and stored in 30% sucrose) in embedding medium such as &#34;Optimal Cutting Temperature compound&#34; (OCT) for a few min in a labeled small petri dish.</li>
		<li>Transfer the tissue in a labeled cryomold containing enough fresh OCT to cover the tissue. Orient the sectioning surface toward the bottom of the cryomold (cross section for the vastus lateralis muscle and sagittal section for the hemi-brain). Avoid formation of bubbles.</li>
		<li>Freeze the samples by placing the cryomold for a few min in a covered foam cooler filled with dry ice, until the OCT turns white. Wrap individual samples in labeled foils, seal them in a plastic bag, and store them at -80 &#176;C overnight or longer.</li>
		<li>Use a cryostat to cut sample sections at a thickness of 10 &#181;m, and mount the sections on the slides. Store the slides at -20 &#176;C, and always keep the slides away from light whenever possible.</li>
	</ol>
	</li>
	<li>Slide preparation
	<ol>
		<li>Remove slides from the freezer and thaw them at room temperature. Cover the tissue section with a drop of mounting media with DAPI. Place a coverslip of appropriate size on top and avoid bubble formation.</li>
		<li>Use nail polish to seal the edges of the coverslip, allow nail polish to dry in the dark at room temperature, and then store the slides in a light-tight container at 4 &#176;C, or proceed with imaging capture of GFP-LC3 puncta.</li>
	</ol>
	</li>
	<li>Quantification of GFP - LC3 puncta by fluorescence microscopy
	<ol>
		<li>Perform epifluorescence microscopy, capturing pictures in the same condition (exposure and settings) for all the samples. For GFP, use an emission wavelength of 525 nm; for DAPI, use 490 nm. Acquire at least ten images per sample using a 60X objective for vastus lateralis and the frontal cortex region of the brain.</li>
		<li>Quantify the number of GFP-LC3 puncta in a tissue area of 2,500 &#181;m2 in each image for statistical analysis, blinded for experimental groups. Calculate the average number from 10 images of each sample, using the automatic measurement function of &#34;Object Count&#34;. Settings for the vastus lateralis muscle are: Threshold L = 10, H = 200; 2X smooth; 1X clean, and settings for the brain frontal cortex are: Threshold L = 11, H = 200; 2X smooth; 1X clean.</li>
	</ol>
	</li>
</ol>
6. Western Blot Analysis on Autophagy Markers
<ol>
	<li>Tissue process
	<ol>
		<li>Prepare the lysis buffer: 50 mM Tris-HCl; 150 mM NaCl; 1 mM EDTA; 1% Triton X-100; protease inhibitor and phosphatase inhibitor (freshly added).</li>
		<li>Remove the tissue of C57BL/6 mice from -80 &#176;C storage. Cut the muscle tissue into small pieces with a razor blade prior to the following process. Add 800 &#181;l (for hemi-brain) or 500 &#181;l (for vastus lateralis) of cold lysis buffer to the sample. Homogenize the sample at 4 &#176;C with a homogenizer at a medium speed.</li>
		<li>Fully lyse the homogenized mixture for another 30 min to 1 hr at 4 &#176;C with rotation.</li>
		<li>Spin down the homogenate at 12,000 x g for 10 min at 4 &#176;C, discard the pellet, and collect the supernatant in a new tube.</li>
	</ol>
	</li>
	<li>Autophagy analysis
	<ol>
		<li>Determine the protein concentration of tissue lysates using the BCA Protein assay kit according to the manufacturer&#39;s specifications. Normalize each sample to the same concentration.</li>
		<li>Combine the sample with the 2x Laemmli sample buffer at a 1:1 ratio, boil it at 95 &#176;C for 5 - 10 min, and proceed with western blot analysis on autophagy markers13,14, such as p62 (anti-p62 antibody, 1:500 dilution) and LC3 (anti-LC3 antibody, 1:500 dilution). Boil the samples immediately after addition of the sample buffer, as longer incubation on ice may generate multiple degraded bands of LC3.</li>
		<li>Quantify p62 and LC3 bands by densitometry analysis using ImageJ, and normalize the value to the corresponding actin band.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

Autophagy is a catabolic process that provides energy and reduces cytotoxicity by lysosomal degradation of cytoplasm components or damaged organelles. Studying autophagy is important to understand the regulation of cellular homeostasis and the mechanisms of stress response. New models and methodologies are emerging in the research field15, to study how impaired autophagy contributes to numerous pathological processes16,17.
<p class="jo...

Autophagy is an evolutionarily conserved degradation pathway, which is induced in response to various stress conditions such as starvation and hypoxia1,2. During autophagy, double-membrane vesicles, called autophagosomes, incorporate unnecessary or damaged subcellular components and transport them into lysosomes for degradation3. Basal autophagy is essential for cellular function and organism development, and impaired basal autophagy is implicated in many disorders, including neurodegeneration, tumorigenesis and type 2 diabetes4,5,6.
The best-known physiological autophagy inducer is starvation. However, it has two major limitations. First, starvation takes a long period to effectively induce autophagy in animals, e.g., 48 hr of food restriction in mice in most organs. Second, starvation barely induces brain autophagy, due to a relatively stable nutrient supply in the brain. In fact, it is also difficult to detect autophagy induction by small-molecule inducers, as many drugs cannot pass the blood brain barrier. Thus, to better analyze the function of autophagy activation in disease pathogenesis, we recently discovered that exercise is a more potent physiological method to induce autophagy in a short period of time7,8,9. Compared with starvation, autophagy is effectively induced by treadmill running as fast as 30 min. Thus, exercise is a convenient and potent physiological approach to study the mechanism of autophagy in mediating health benefits and preventing diseases.
There are several protein markers for the detection of autophagy activity, including LC3 and p62. LC3 (microtubule-associated protein 1A/1B-light chain 3) is a cytosolic protein (LC3-I form) that is conjugated to PE (phosphatidylethanolamine) upon autophagy induction. PE-lipidated LC3 (LC3-II form) is recruited onto autophagosomal membranes and can be used to visualize autophagosomes when labeled with GFP. Its translocation from the cytosol to punctate structures of autophagosomes under microscopy is an indication of autophagy induction. p62 is a cargo receptor for autophagy substrates (such as ubiquitination proteins), and is incorporated into autophagosomes as well. Since the protein is degraded in lysosomes along with autophagosomes, its levels can be used to measure the autophagy flux. Here we show how to use these markers to quantify autophagy in different mouse tissues induced by aerobic exercise, including forced exercise (treadmill) and voluntary exercise (running wheels). The same procedures can also be applied to in vivo measurement of autophagy after treatment of other inducers.

<table border="0" cellpadding="0" cellspacing="0" width="863">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Treadmill</td>
			<td style="width:204px;">Columbus Instruments</td>
			<td style="width:195px;">150-RM Exer 3/6</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mouse running wheel</td>
			<td style="width:204px;">Super Pet</td>
			<td style="width:195px;">100079365</td>
			<td>diameter 11.4 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Odometer</td>
			<td style="width:204px;">Bell</td>
			<td style="width:195px;">DASHBOARD 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Syringe pump</td>
			<td style="width:204px;">KD Scientific</td>
			<td style="width:195px;">KDS100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Fluorescence microscope</td>
			<td style="width:204px;">Nikon</td>
			<td style="width:195px;">Model: inverted microscope ECLIPSE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Cryostat</td>
			<td style="width:204px;">Leica</td>
			<td style="width:195px;">CM 1850UV</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Homogenizer</td>
			<td style="width:204px;">IKA</td>
			<td style="width:195px;">003737001 / Model: T10 Basic S1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Chloroquine</td>
			<td style="width:204px;">CAYMAN CHEMICAL COMPANY</td>
			<td style="width:195px;">14194</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Parafolmaldehye</td>
			<td style="width:204px;">SIGMA-ALDRICH</td>
			<td style="width:195px;">P6148</td>
			<td>Personal protection equipment required. This product may release formaldehyde gas, a chemical known to cause cancer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mounting media</td>
			<td style="width:204px;">Vector Laboratories</td>
			<td style="width:195px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">p62 antibody</td>
			<td style="width:204px;">BD Biosciences</td>
			<td style="width:195px;">610833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">LC3 antibody</td>
			<td style="width:204px;">Novus Biologicals</td>
			<td style="width:195px;">NB100-2220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2X Laemmli Sample Buffer</td>
			<td style="width:204px;">Bio-Rad Laboratories</td>
			<td style="width:195px;">161-0737</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">ImageJ</td>
			<td style="width:204px;">NIH</td>
			<td style="width:195px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

activating autophagy by aerobic exercise in mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

This protocol describes two different methods to induce autophagy in mouse tissues by aerobic exercise: a total of 90 min of forced exercise on a multi-lane treadmill proceeded by two days of acclimation; or two weeks of voluntary exercise on a running wheel used by single-housed mice. In each exercise protocol, we can measure the autophagy flux by fluorescence microscopy and western blot analysis in various organs.
We used a tr...

Watch this Scientific Journal Video about Activating Autophagy by Aerobic Exercise in Mice at JoVE.com

Activating Autophagy by Aerobic Exercise in Mice

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

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Research

JoVE Journal

Biology

11.7K Views.  Northwestern University. Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

Video: Activating Autophagy by Aerobic Exercise in Mice

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary.
Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of variability among studies. Video publication is therefore useful in this case, and we demonstrate low-variability, high-quality results.

This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

Determining the Contribution of the Energy Systems During Exercise

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given physical activity. Although some sports are relatively easy to be reproduced in a laboratory (e.g., running and cycling), a number of sports are much more difficult to be reproduced and studied in controlled situations. This method presents how to assess the differential contribution of the energy systems in sports that are difficult to mimic in controlled laboratory conditions. The concepts shown here can be adapted to virtually any sport.
The following physiologic variables will be needed: rest oxygen consumption, exercise oxygen consumption, post-exercise oxygen consumption, rest plasma lactate concentration and post-exercise plasma peak lactate. To calculate the contribution of the aerobic metabolism, you will need the oxygen consumption at rest and during the exercise. By using the trapezoidal method, calculate the area under the curve of oxygen consumption during exercise, subtracting the area corresponding to the rest oxygen consumption. To calculate the contribution of the alactic anaerobic metabolism, the post-exercise oxygen consumption curve has to be adjusted to a mono or a bi-exponential model (chosen by the one that best fits). Then, use the terms of the fitted equation to calculate anaerobic alactic metabolism, as follows: ATP-CP metabolism = A1 (mL . s-1) x t1 (s). Finally, to calculate the contribution of the lactic anaerobic system, multiply peak plasma lactate by 3 and by the athlete&#x2019;s body mass (the result in mL is then converted to L and into kJ).
The method can be used for both continuous and intermittent exercise. This is a very interesting approach as it can be adapted to exercises and sports that are difficult to be mimicked in controlled environments. Also, this is the only available method capable of distinguishing the contribution of three different energy systems. Thus, the method allows the study of sports with great similarity to real situations, providing desirable ecological validity to the study.

This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional microscopy to surface nephron features, and 2-photon studies are limited in that interventions can only be assessed at the organ, rather than the nephron level. In particular, micropuncture studies of the glomeruli of mice have been challenged by the paucity of surface glomeruli in mice. To address this limitation in order to pursue studies of aspirate from Bowman&#39;s space in mouse physiologic models, we developed 2-photon glomerular micropuncture. We present a novel surgical preparation that allows lateral access to the kidney while preserving the required vertical imaging column for 2-photon microscopy. Administration of high molecular weight fluorescein isothiocyanate (FITC)-dextran is used to render the renal vasculature and therefore glomeruli visible for 2-photon imaging. A quantum dot-coated pipette is then introduced under stereotactic guidance to a glomerulus selected from the several to many which may be visualized within the imaging window. In this protocol, we provide details of the preparation, materials, and methods necessary to carry out the procedure. This technique facilitates previously-impossible physiologic study of the kidney, including recovery of filtrate from Bowman&#39;s space and all segments of the nephron within the imaging depth limit, about 100 &#181;m below the renal capsule. Pressure, charge and flow may all be measured using the introduced pipette. Here, we provide representative data from liquid chromatography/mass spectrometry performed on aspirate from Bowman&#39;s space. We expect this technique to have wide applicability in renal physiologic investigation.

We present use of 2-photon microscopy to place a micropipette within Bowman&#39;s urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional ...