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.

주 :. 변성 침전 법을 이용하여 엑소 좀 뇨의 분리에 관련된 단계는도 1에 도시되는 초기 단계 원심 분리에 의해 큰 사균의 제거 및 세포 파편을 포함한다. 각각의 후속 단계에서 수집 된 상층 액에서 얻은 최종 펠렛은 단백질, RNA 및 DNA의 분리를위한 추가의 추출 용액에 용해되는 엑소 좀에 대응한다. 소변 1. 컬렉션 <ol><li> 소변 수집하기 전에, 제조업체의 지시에 따라 단백질 분해 효소 억제제 칵테일을 준비하고 빈 멸균 용기에이 칵테일의 3.125를 가하여. 프로테아제 저해제 칵테일을 포함하는 튜브이어서 소변 수집 지원자에게 분배 될 수있다. 주 : 프로테아제 억제제를 첨가하는 이유는 소변에서 단백질 분해를 최소화하는 것이다. 우리는 특별히에서 프로테아제 억제제 칵테일을 사용하지만시그마, 다른 시판 프로테아제 억제제가 치환 될 수있다. </li><li> 처음 무효 소변의 약 10 ml의를 수집합니다. 즉시 수집 후 저장 또는 냉동하지 않고 소변을 처리합니다. 수정 된 침전 법을 사용하여 중복에 소변 엑소 좀을 분리; 다른 비뇨기 엑소 입자의 정량화 동안 중복 중 하나는, DNA, RNA 및 총 단백질 추출을위한 것이다. 참고 : 24 시간 소변 시료를 4 ℃에서 저장하여 엑소의 격리를 위해 수집 할 수 있습니다. </li></ol> 세포 파편과 탬-Horsfall 단백질 2. 제거 (THP) <ol><li> 15 ml의 원심 분리기 튜브에 소변 샘플을 전송합니다. 원심 분리기 37 ° C에서 10 분 동안 17,000 XG에 소변 10ml의 세포 및 세포 찌꺼기 (도 1)를 제거한다. </li><li> 피펫을 사용하여, 추가 처리를 위해 새로운 튜브에 뜨는을 전송합니다. 차단 용액 500 μL의 펠렛을 재현 탁 (250 mM의 크로즈, 10 밀리미터 하시 느니라최종 농도 200 ㎎ / ㎖)에서 37 ° C : DL-디티 오 트레이 톨 (DTT 10 분 인큐베이션 anolamine, pH를 7.6). 참고 : DTT는 수확량 감소로 이어지는, 엑소 좀 트랩 THP를 저하 추가됩니다. 뜨는 전송하는 동안 방해받지 펠렛을 떠날주의, 상층 액은 펠릿이 없는지 확인합니다. </li><li> 소용돌이 펠렛 시료 펠릿까지 매 2 분을 완전히 용해시킨다. 그런 다음 용해 펠릿에 격리 솔루션의 500 μl를 추가합니다. </li><li> 즉시 37 ℃에서 10 분 동안 17,000 XG에 분리 펠릿 용액, 원심 첨가 한 후. 피펫은 뜨는 및 이전 버전 수집 된 상층 액과 결합. </li><li> SDS-PAGE 분석을위한 격리 솔루션의 50 μL에 펠렛을 재현 탁. 주 : TBS 또는 PBS 펠릿을 재현 탁 대신 분리가 사용될 수도있다. </li></ol> 엑소 펠렛 3. 분리 <ol><li> 수집 된 supern 11 히드로atant, ExoQuick-TC 시약 3.3 ml에 추가하고 적어도 12 시간 동안 4 ° C에서 배양한다. 주 : TC-ExoQuick 시약을 상층 액 부피의 비는 증가 또는 exosomal 수율을 감소하도록 조정될 수있다. 우리는 추가 시약의 낮은 볼륨 엑소 좀 작은 수율을 생산하는 것을 관찰했다. </li><li> 부화 후, 원심 분리기 25 ° C에서 30 분 동안 10,000 XG에 샘플 / ExoQuick-TC 믹스. </li><li> SDS-PAGE 분석을위한 새로운 튜브에 뜨는을 전송합니다. </li><li> 엑소 좀 펠렛은 DNA, RNA 및 단백질 추출 및 CD9 11 ELISA를 이용한 엑소 입자의 정량화 -80 ° C에서 저장 될 수있다. 참고 : 엑소 펠릿이 제한 동결 융해와 1 년 동안 -80 ° C에 저장할 수 있습니다. </li></ol> 4. 바이오 마커 검출 및 엑소 펠렛의 분석 <ol><li> 하류 분석에서, DNA / RNA / 단백질 단리 키트 및 RNA 분리 KI를 사용하여 엑소 좀 펠렛으로부터 DNA, RNA 및 단백질을 추출제조업체의 지침에 따라 t. 샘플 2 μL의 260 nm와 280 nm에서의 흡광도를 측정함으로써 Nanodrop 분광 광도계를 이용하여 농도를 결정한다. </li><li> 정량 실시간 RT-PCR (qPCR에)의 TaqMan 마이크로 RNA 분석을 사용하여이 또는 miRNA에 특이 적 프라이머 (우리의 경우, 우리는 인간은 miR-192과의 miR-1207-5p 별 분석을 사용) 수행합니다. </li><li> SDS-PAGE 분석을 위해 일차원 전기 영동을 4 내지 12 % 비스 -Tris 겔 분리 단백질 시료를 사용한다. 제조업체의 지시에 따라 실버 염색 키트를 사용하여 젤을 가시화. </li><li> 제조업체의 지시 사항에 따라 PVDF 멤브레인을 사용하여 웨스턴 블롯 분석을 수행합니다. 웨스턴 블롯 검출 종양 감수성 유전자 (101) 단백질 또는 TSG101에 세포 사멸 연결된 유전자-2 상호 작용하는 단백질 X 또는 ALIX와 마우스 단일 클론 항체에 토끼 폴리 클로 날 항체를 사용합니다. 오픈 액세스 NIH ImageJ에 소프트웨어에 의해 밴드의 밀도를 정량화 (http://rsbweb.nih.gov/ij/) 또는유사한 프로그램. </li><li> 제조업체의 지시에 따라, CD9 ELISA 키트를 사용하여 얻어진 뇨 엑소 입자의 수​​를 정량화. </li></ol>

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이 기사에 대한 출판 비용은 SBI 생명 과학에 의해 지불했다.

소변에서 엑소 좀 침전을 포함하는 대부분의 방법은 탬-Horsfall 단백질 (THP)에 의해 형성되는 고분자 네트워크의 제거를 해결하지 않습니다. 이 단백질은 추정되는 트랩 초기 저속 원심 엑소 좀 동안 소변에 다량으로 존재한다. 수정 된 방법에서 우리는 엑소 침전하기 전에 DTT를 사용하여 THP를 감소시켰다. 도 2에 도시 된 바와 같이, THP 더 많은 양이 단백질의 제거를 나타내는, 최종 상?...

소변 엑소 좀은 miRNA가 1 농축 한 것으로 나타났습니다 소변 배수 시스템을 긋고 사구체 족 세포, 신장 세뇨관 세포와 세포를 포함 비뇨기 공간에서 정상 및 병적 인 상태에서 모든 종류의 세포에서 파생 된 multivesicular 기관 (MVB)의 100 나노 미터의 작은 내부 소포 있습니다 -2. 많은 연구자들은 건강한 사람의 소변뿐만 아니라 신장 및 / 또는 체계적인 질병에 3-5 가진 사람에서 분리 된 엑소 좀에 별개의 단백질을 발견했다. 엑소 좀은 8-9 초 원심 분리 또는 침전에 10-11 nanomembrane 필터를 조합, 예컨대 수크로오스 또는 6-7없이 두 단계 차동 초 원심 분리 등의 다양한 방법을 이용하여 분리 할 수있다. 우리는 최근에 소변 엑소 좀을 격리하며 miRNA의 단백질 바이오 마커 (11)를 검출하는, 간단하고, 빠르고, 확장 가능하고 효과적인 방법을 개발했다. 바이오 마커는 다양한 생물학적 다양한 유체에서 발견 될 수 있지만, UR오프라인은 비교적 비 침습적 방식으로 다량으로 얻을 수 있기 때문에 신장 및 요로 질환 환자를위한 가장 편리한 선택이다. 소변 엑소 좀을 6-11을 분리하는 방법은 여러 가지가 있지만, 가장 일반적으로 사용되는 절차는 30 % 자당 쿠션 차동 초 원심 분리에 따라 달라집니다. 이 방법은 효율적이며,이 절차와 절연 얻어진 엑소 좀의 품질이 높은 반면, 기술 자체는 숙련 된 사람이 필요하고 시간이 많이 소요되는 여러 단계를 초 원심 분리에 필요하다. 여과 및 침전 등의 다른 방법은, 독점적 인 침전 시약을 사용하는 것을 포함하여, 엑소 좀의 분리를 위해 개발 된 (즉, ExoQuick-TC : 시스템 생명 과학, 마운틴 뷰, CA). 이 시약을 사용하여 침전 빠르고 비교적 쉽지만, 절연 엑소 좀의 순도는 초 원심 메트 비해 낮다OD. 최근 우리는 우리 실험실에서 개발 ExoQuick 11-TC를 사용하여 침전 법의 변형을 포함 비뇨기 엑소 좀의 분리를위한 여섯 방법을 비교 하였다. 우리는이 수정 침전 법은 단백질, miRNA의, mRNA를 자체 빠르고 초 원심보다 쉽게​​ 구현했다 절차의 높은 수량을 산출 한 것으로 나타났습니다. 여기, 우리는 단계적으로 수정 된 침전 법의 실험 프로토콜을 설명하고 엑소 분리하는 다른 방법에 비해이 기술의 장점과 단점에 대한 논의를 포함한다. 변성 침전 법은 exosomal 단백질과 상관하고, 소변 엑소 수율을 감소하는 것으로 알려져 탬-Horsfall 단백질 제거하여 엑소 입자의 초기 침전을 포함한다. 우리는 이러한 수정은 소변에서 exosomal 준비의 수율과 순도를 증가 것을 발견했다.

<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.

소변 엑소 좀은 신장 상피 세포에서 분비되는 단백질의 miRNA와 mRNA의 바이오 마커를 수행한다. 이러한 엑소 좀의 분리 및 검출은 당뇨병 성 신증으로 신장 질환의 조기 발견을위한 유용한 진단 도구를 제공 할 수있다. 인간을 대상으로 수집 된 소변 샘플에서 엑소 좀의 분리를위한 몇 가지 방법이 있습니다. 우리는 이전에 소변 엑소 분리의 여섯 방법을 비교하여 단백질의 mRNA와 miRNA의 수준 (11)...

Watch this Scientific Journal Video about A Modified Precipitation Method to Isolate Urinary Exosomes at JoVE.com

A Modified Precipitation Method to Isolate Urinary Exosomes

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

수정 된 침전 법은 요도 엑소 좀을 분리

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

a-modified-precipitation-method-to-isolate-urinary-exosomes

Research

JoVE Journal

Medicine

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

비디오: 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.

노화 암 세포를 정량화하는 민감한 방법

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

연구

의학

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.

수정 커프 기술을 사용하여 쥐의 자궁 심장 이식 모델

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.

쥐의 방광 통증 모델로 방광 팽창 유발 내장 운동 응답

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.

이소성 마우스 심장 장기 이식에 대한 수정 방법

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.

하부 요로 약리학을 평가하는 방법으로 방광 평활근 스트립 수축성

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.

수정 된 버그 스트롬 기술을 이용하여 인간의 골격 근육 생검 절차

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

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.

그대로 Glomeruli 성인 쥐 신장에서 분리 하는 효율적인 체질 하 방법

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).

요로 기능 검사를 위한 성인 남성 요로의 초음파 검사

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.

생쥐의 심근경색 유도를 위한 수정된 간단한 방법

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

A Modified Vessel-Sparing Microsurgical Vasoepididymostomy

Microsurgical vasoepididymostomy (MVE) is the main surgical treatment for epididymal obstruction. The vasal vessels are ligated during MVE. However, preserving the vasal vessels during MVE might better simulate the normal physiological structure and be meaningful for patients who have undergone varicocelectomy. Nevertheless, preserving the vasal vessels might elevate the risk of increasing the tension of anastomosis, affecting the patency rate and leading to delayed postoperative bleeding. Therefore, we developed a novel vessel-sparing MVE to make it safer. Here is a summary of the improvements to the procedure. 1) The retrograde dissociation of the vasal vessels on the proximal testicular side was adopted as the main method, and the anterograde dissociation of the vasal vessels on the distal testicular side was adopted as a supplement to dissociate the vasal vessels to be preserved. This improvement ensures the blood supply to the vas deferens that will be used for anastomosis and also provides longer vasal vessels, which reduces the tension of anastomosis. 2) By fixing the vas deferens to be anastomosed and the broken end of the vas deferens, the free vasal vessels get fixed, which resolves the problem of transmission of vas tension to the vasal vessels and reduces the risk of vasal vessel hemorrhage. 3) Dissociation of the vas deferens after opening the tunica vaginalis increases the mobilization of the vas deferens, which also makes the new procedure easier to complete. The evaluation of the outcomes of this new procedure showed that no significant postoperative complications occurred in the patients, and the patency rate was no different from that of the conventional procedure. Therefore, this new, improved procedure can be considered safe, with satisfactory postoperative results.

Here, we present a protocol to preserve the vasal vessels in microsurgical vasoepididymostomy. The surgical security is enhanced by preserving the vasal vessels using a retrograde-anterograde dissociation and fixing the vasal vessels.

변형된 혈관 보존 미세 외과적 혈관절개술

Microsurgical vasoepididymostomy (MVE) is the main surgical treatment for epididymal obstruction. The vasal vessels are ligated during MVE. However, ...