We are grateful to the Roney Family Foundation for the support of this study. We acknowledge the contribution of Dr. M. Lucrecia Alvarez to the planning and execution of the experiments described in this work.

ПРИМЕЧАНИЕ:. Этапы выделения мочевой экзосом с использованием модифицированного метода осаждения показаны на рисунке 1 Первые шаги включают удаление больших мертвых клеток и клеточных обломков путем центрифугирования. Конечный осадок, полученный из надосадочной жидкости, полученной от каждого последующего шага соответствует экзосома, который растворен в растворе изоляции для дальнейшей экстракции белка, РНК и ДНК. 1. Сбор мочи <ol><li> До сбора мочи, подготовить ингибитор протеазы коктейль в соответствии с инструкциями изготовителя и добавить 3,125 мл этого коктейля на пустой стерильной емкости. Трубка, содержащая ингибитор протеазы коктейль затем могут быть распространены на волонтеров для сбора мочи. Примечание: причина для добавления ингибитора протеазы, чтобы минимизировать деградацию белка в образце мочи. Хотя мы специально использовали ингибитор протеазы коктейль изСигма, другие коммерчески доступные ингибиторы протеазы могут быть замещены. </li><li> Собирают приблизительно 10 мл первого пустот мочи. Процесс мочу сразу после сбора без хранения или охлаждения. Изолировать мочевых экзосомы в двух экземплярах с использованием модифицированного метода осаждения; один из дубликатов для ДНК, РНК и общего белка экстракцией, а другой для количественного определения частиц экзосом мочевых. Примечание: 24 ч образец мочи можно собрать для изоляции экзосома при хранении при 4 ° С. </li></ol> 2. Удаление остатков клеток и Тамма-Horsfall белка (THP) <ol><li> Трансфер образцы мочи до 15 мл центрифужные пробирки. Центрифуга 10 мл мочи при 17000 мкг в течение 10 мин при 37 ° С, чтобы устранить клетки и клеточных обломков (рисунок 1). </li><li> С помощью пипетки, передавать супернатант в чистую пробирку для дальнейшей обработки. Ресуспендируют осадок в 500 мкл раствора изоляции (250 мМ сахарозы, 10 мМ испытующемуanolamine, рН 7,6) с последующим 10-минутной инкубации с DL-дитиотреитола (DTT: конечная концентрация 200 мг / мл) при 37 ° С. Примечание: ДТТ добавляют к ухудшению ТНР, который поглощает экзосомы, что приводит к снижению урожайности. Позаботьтесь, чтобы оставить осадок ненарушенных во время передачи супернатанта и убедитесь, что супернатант ясно окатышей. </li><li> Образцы гранул Вихревые каждые 2 мин до осадок полностью не растворится. Затем добавляют 500 мкл раствора изоляции в растворенном осадке. </li><li> Сразу же после добавления изоляции раствора к осадку, центрифугируют при 17000 х г в течение 10 мин при 37 ° С. Пипеткой супернатант и в сочетании с надосадочной жидкости, собранной ранее. </li><li> Ресуспендируют осадок в 50 мкл изоляции раствора для анализа SDS-PAGE. Примечание: PBS, или терапию, может быть использован вместо раствора изоляции, чтобы вновь суспендируют таблетку. </li></ol> 3. Выделение экзосома Пелле <ol><li> Для 11 мл собранной supernatant, добавить 3,3 мл ExoQuick-TC реагента и инкубировать при температуре 4 ° С в течение по крайней мере 12 часов. Примечание: Отношение объема надосадочной жидкости до ExoQuick-TC реагента можно регулировать, чтобы увеличить или уменьшить выход exosomal. Мы заметили, что меньшие объемы добавленной реагента производят меньшие урожаи экзосом. </li><li> После инкубации, центрифуги смеси образца / ExoQuick-ТК при 10000 х г в течение 30 мин при 25 ° С. </li><li> Передача супернатант в новую пробирку для анализа SDS-PAGE. </li><li> Экзосом осадок можно хранить при -80 ° С в течение ДНК, РНК и белка экстракции и количественного определения частиц экзосом использованием ELISA CD9 11. ПРИМЕЧАНИЕ: экзосом осадок может храниться при -80 ° С в течение 1 года с ограниченным замораживания и оттаивания. </li></ol> 4. биомаркеров для выявления и анализа экзосома Пелле <ol><li> Для последующих анализов, извлечения ДНК, РНК и белка из экзосома гранул с использованием ДНК / РНК / комплект изоляции белка и РНК изоляции кит в соответствии с инструкциями изготовителя. Определение концентрации с использованием спектрофотометра NanoDrop путем измерения оптической плотности при 260 и 280 нм с 2 мкл образца. </li><li> Выполните количественный реального времени RT-PCR (КПЦР) с помощью TaqMan микроРНК анализов (в нашем случае мы использовали человеческие микроРНК-192 стран и Мир-1207-5p специфические анализы) или микроРНК-специфических праймеров. </li><li> Для анализа SDS-PAGE, используйте 4-12% Bis-Tris гель и отдельные образцы белка одномерного электрофореза. Представьте гели с использованием набора окраски серебром в соответствии с инструкциями изготовителя. </li><li> Выполните Вестерн-блоттинга с использованием PVDF мембраны в соответствии с указаниями производителя. Использование кроличьих поликлональных антител к апоптоз-сшитый ген-2 взаимодействующего белка X или ALIX и мышиных моноклональных антител к ген восприимчивости опухолей 101 белка или TSG101 в Вестерн-блот-обнаружения. Количественная плотность полос открытого доступа программного обеспечения NIH ImageJ (http://rsbweb.nih.gov/ij/) илиАналогичная программа. </li><li> Определить число мочевыводящих частиц экзосом полученных с использованием набора CD9 ELISA в соответствии с инструкциями изготовителя. </li></ol>

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Публикация сборы для этой статьи были оплачены SBI биологических наук.

Большинство методов с участием осаждение экзосом из мочи не учитывают удаление полимерного сети, образованной Тамм-Хорсфолл белка (ТНР). Этот белок присутствует в больших количествах в моче, где предположительно ловушки экзосомы во время начальной низкоскоростным центрифугированием...

Мочевой экзосомы небольшие внутренние пузырьки 100 нм мультивезикулярных органов (MVB), полученных из клеток всех типов в нормальных и патологических условиях в моче пространства, включая клубочков подоцитов, клетках почечных канальцев и клеток, выстилающих мочевые системы дренажа, которые появляются в обогащении микроРНК 1 -2. Многие исследователи обнаружили различные белки в экзосом, выделенных из мочи здоровых лиц, а также пациентов с почечной и / или системными заболеваниями 3-5. Экзосомы может быть выделен с помощью различных методов, таких как двухступенчатой ​​дифференциального ультрацентрифугирования с или без сахарозы 6-7, комбинируя наномембраны фильтр ультрацентрифугирования 8-9 или 10-11 осадков. Мы недавно разработали простой, быстрый, масштабируемый и эффективный метод, чтобы изолировать мочевых экзосомы и выявления микроРНК и белка биомаркеров 11. Хотя биомаркеры могут быть обнаружены в самых разных биологических жидкостях, игине является наиболее удобен для больных с заболеваниями почек и мочевого тракта, поскольку он может быть получен в больших количествах в относительно неинвазивным способом. Хотя существует несколько методов, чтобы изолировать мочевого экзосомы 6-11, наиболее часто используемые процедуры, зависит от дифференциального ультрацентрифугирования с 30% сахарозы подушке. В то время как этот способ является эффективным и качество получаемых экзосом изолированных с этой процедурой является высокой, сама техника требует квалифицированного персонала и отнимает много времени и требует несколько шагов ультрацентрифугирования. Другие методы, такие как фильтрация и осадков, были разработаны для изоляции экзосом, в том числе тот, который использует запатентованную осадков реагент (т.е. ExoQuick-TC: Система биологических наук; Mountain View, CA). Хотя осадков использованием этого реагента очень быстро и сравнительно легко, чистота выделенных экзосом является низкой по сравнению с ультрацентрифугирования метOD. Мы недавно сравнил шесть методов для выделения в моче экзосом, в том числе модификации метода осаждения с использованием ExoQuick-TC, которые мы разработали в нашей лаборатории 11. Мы обнаружили, что этот модифицированный способ осадков дали более высокие количества белка, микроРНК, и мРНК и сама процедура была быстрее и проще в реализации, чем ультрацентрифугированием. Здесь мы опишем экспериментальный протокол нашего модифицированного метода осаждения ступенчато, и включают в себя обсуждение преимуществ и недостатков этого метода по сравнению с другими методами изоляции экзосома. Модифицированный метод осаждения включает начальное осаждение частиц экзосом, с последующим удалением Тамм-Хорсфолл белка, который коррелирует с exosomal белков, и известно, снижают экзосома выход из мочи. Мы обнаружили, что эти модификации повышает выход и чистоту препарата exosomal из мочи.

<table border="0" cellpadding="0" cellspacing="0" width="736">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:111px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:291px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Corning 15ml Centrifuge Tube</td>
			<td style="width:111px;">Corning (Sigma Aldrich)</td>
			<td style="width:119px;">CLS430791</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protease Inhibitor Cocktail</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:111px;">Sorvall</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DL-dithiothreitol</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:119px;">D9779</td>
			<td>used at final concentration of 200 mg/ml</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Novex NuPAGE SDS-PAGE system (4-12 % Bis-Tris Gel 1.0 mm, 10 well</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:119px;">NP0321BOX</td>
			<td>For SDS-PAGE analysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ECL Advance Western blotting detection kit</td>
			<td style="width:111px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">RPN2135</td>
			<td>For western blot detection of biomarkers</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Exoquick-TC Reagent</td>
			<td style="width:111px;">System Biosciences (SBI)</td>
			<td style="width:119px;">EXOTC50A-1</td>
			<td>For exosome isolation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Exosome CD9 ELISA Complete Kit</td>
			<td style="width:111px;">System Biosciences (SBI)</td>
			<td style="width:119px;">EXOEL-CD9-A-1</td>
			<td>For exosome quantification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">AllPrep DNA/RNA/Protein mini kit</td>
			<td style="width:111px;">Qiagen</td>
			<td style="width:119px;">80004</td>
			<td>For DNA, RNA, Protein isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rneasy MinElute Cleanup kit</td>
			<td style="width:111px;">Qiagen</td>
			<td style="width:119px;">74204</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NanoDrop 2000 UV-Vis Spectrophotometer</td>
			<td style="width:111px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>For Protein Quantification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ProteoSilver Sliver Stain Kit</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:119px;">PROTSIL1-1KT</td>
			<td>For staining SDS-PAGE gels</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Xcell SureLock Mini-Cell Electrophoresis System</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td>For running the SDS-PAGE gels</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan microRNA assays</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td>For RT-PCR assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">qBasePlus v1.5 software</td>
			<td style="width:111px;">Biogazelle NV</td>
			<td style="width:119px;"></td>
			<td>For analysis of RT-PCR assay data</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rabbit polyclonal antibody to ALIX</td>
			<td style="width:111px;">Millipore</td>
			<td style="width:119px;"></td>
			<td>For western blot detection of biomarkers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mouse monoclonal antibody to TSG101</td>
			<td style="width:111px;">Abcam</td>
			<td style="width:119px;"></td>
			<td>For western blot detection of biomarkers</td>
		</tr>
	</tbody>
</table>

a modified precipitation method to isolate urinary exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

Мочевой экзосомы нести белок, микроРНК и мРНК биомаркеров, выделяемые из почек эпителиальных клеток. Выделение и определение из этих экзосом может обеспечить полезную диагностическую инструмент для ранней диагностики заболеваний почек, таких как диабетическая нефропатия. Есть неско...

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A Modified Precipitation Method to Isolate Urinary Exosomes

This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

Модифицированный Осадки Метод изолировать мочевых экзосомы

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. ...

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Research

JoVE Journal

Medicine

17.5K Views.  Translational Genomics Research Institute (TGen). This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

Video: A Modified Precipitation Method to Isolate Urinary Exosomes

A Sensitive Method to Quantify Senescent Cancer Cells

Human cells do not indefinitely proliferate. Upon external and/or intrinsic cues, cells might die or enter a stable cell cycle arrest called senescence. Several cellular mechanisms, such as telomere shortening and abnormal expression of mitogenic oncogenes, have been shown to cause senescence. Senescence is not restricted to normal cells; cancer cells have also been reported to senesce.
	Chemotherapeutical drugs have been shown to induce senescence in cancer cells. However, it remains controversial whether senescence prevents or promotes tumorigenesis. As it might eventually be patient-specific, a rapid and sensitive method to assess senescence in cancer cell will soon be required.
	To this end, the standard &beta;-galactosidase assay, the currently used method, presents major drawbacks: it is time consuming and not sensitive. We propose here a flow cytometry-based assay to study senescence on live cells. This assay offers the advantage of being rapid, sensitive, and can be coupled to the immunolabeling of various cellular markers.

Whether senescence prevents or promotes tumorigenesis remains controversial. Since chemotherapeutical drugs can induce cancer cells to senesce, studying senescence is essential for proposing new therapies. However, the standard and broadly used &beta;-galactosidase assay presents major drawbacks. We propose here a rapid and sensitive flow cytometry-based assay to quantify senescence.

Чувствительным методом для количественной стареющие клетки рака

Human cells do not indefinitely proliferate. Upon external and/or intrinsic  cues, cells might die or enter a stable cell cycle arrest called senescence. ...

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Мышиный шейки Трансплантация сердца Модель с использованием модифицированного манжеты Техника

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Мочевой пузырь Вздутие Вызванные висцеромоторный Ответы в качестве модели для мочевого пузыря боли у мышей

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

A Modified Method for Heterotopic Mouse Heart Transplantion

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents available. A difficulty is presented due to the small size of the animal and the considerable technical challenges of the microsurgery involved in heart transplantation. In particular, a high rate of technical failure early after transplantation may result from recipient death and post-operative complications such as hind limb paralysis or a non-beating heart. Here, the complete technique for heterotopic mouse heart transplantation is demonstrated, involving harvesting the donor heart and its subsequent implantation into a recipient mouse. The donor heart is harvested immediately following in situ perfusion with cold heparinized saline and transection of the ascending aorta and pulmonary artery. The recipient operation involves preparation of the abdominal aorta and inferior vena cava (IVC), followed by end-to-side anastomosis of the donor aorta with the recipient aorta using a single running 10-0 microsuture and a similar anastomosis of the donor pulmonary artery with the recipient IVC. Following the operation the animal is injected with 0.6 ml normal saline subcutaneously and allowed to recover on a 37&#160;&#176;C heating pad. The results from 227 mouse heart transplants are summarized with a success rate at 48 hr of 86.8%. Of the 13.2% failures within 48 hr, 5 (2.2%) experienced hind limb paralysis, 10 (4.4%) had a non-beating heart due to graft ischemic injury and/or thrombosis, while 15 (6.6%) died within 48 hr.

A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis.

Модифицированный метод для гетеротопической Mouse сердца Transplantion

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents ...

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.

This manuscript presents a simple, yet powerful, in vitro method for evaluating smooth muscle contractility in response to pharmacological agents or nerve stimulation. Main applications are drug screening and understanding tissue physiology, pharmacology,&#160;and pathology.

Гладких мышц мочевого пузыря Газа Сократимость как метод оценки нижних мочевых путей фармакология

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&amp;#246;m Technique

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

Человеческого скелета Процедуры биопсии мышц с использованием модифицированного Бергстром Техника

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Выделение и профилирование микроРНК содержащих экзосомы от человека Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Эффективный метод просеивания изолировать нетронутыми клубочков из почек взрослых крыс

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting in a significant economic and quality of life burden. Transgenic and other mouse models have been developed to recreate various aspects of this multifactorial disease; however, methods to accurately quantitate urinary dysfunction and the effectiveness of new therapeutic options are lacking. Here, we describe a method that can be used to measure bladder volume and detrusor wall thickness, urinary velocity, void volume and void duration, and urethral diameter. This would allow for the evaluation of disease progression and treatment efficacy over time. Mice were anesthetized with isoflurane, and the bladder was visualized by ultrasound. For non-contrast imaging, a 3D image was taken of the bladder to calculate volume and evaluate shape; the bladder wall thickness was measured from this image. For contrast-enhanced imaging, a catheter was placed through the dome of the bladder using a 27-gauge needle connected to a syringe by PE50 tubing. A bolus of 0.5 mL of contrast was infused into the bladder until a urination event occurred. Urethral diameter was determined at the point of the Doppler velocity sample window during the first voiding event. Velocity was measured for each subsequent event yielding a flow rate. In conclusion, high frequency ultrasound proved to be an effective method for assessing bladder and urethral measurements during urinary function in mice. This technique may be useful in the assessment of novel therapies for BPH/LUTS in an experimental setting.

We describe the use of high frequency ultrasound with contrast imaging as a method to measure bladder volume, bladder wall thickness, urine velocity, void volume, void duration, and urethral diameter. This strategy can be used to assess voiding dysfunction and treatment efficacy in various mouse models of lower urinary tract dysfunction (LUTD).

Ультрасонография взрослых мужских мочевыводящих путей для функционального тестирования мочи

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting ...

A Modified Simple Method for Induction of Myocardial Infarction in Mice

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging. The current study describes a simple and efficient process for induction of MI in mice with readily available materials. The mouse chest skin was cut open under stable anesthesia. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an MI model can be established in mice within 2-3 min with readily available materials.

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

Модифицированный простой метод индукции инфаркта миокарда у мышей

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...