이러한 연구 GAH에 NIH R01 AI36532에 의해 기금에 의해 부분적으로 지원되었다. 우리는 박사 Tibor Nadasdy에서 제공하는 이식 병리학의 뛰어난 지원을 인정하고,이 연구 노력을 시작으로 후반 박사 찰스 G. Orosz에서 제공하는 귀중한지도 데다가 것입니다.

기증자 장기 적출 수술 (2) : <ol><li> 65mg/kg pentobarbital IP를 사용하여 기증자를 마취, 마취는 Isoflurane (2-3 %)에 의해 유지됩니다. </li><li> 기증자 복부를 입력하고 오른쪽 옆으로 소장 이동하여 왼쪽 신장을 폭로 가슴에서 치골에 정중선 절개를합니다. </li><li> 8-0 실크 봉합사와 부신과 고환 혈관 ligating과 나누어 왼쪽 신장을 분리. </li><li> 신장 동맥과 정맥과의 접합 위의 대동맥을 준비 시켜라. </li><li> 신장 동맥과 정맥과의 접합부 위 아래 하부 대정 맥 (IVC)을 동원. </li><li> 출혈을 최소화하기 위해 8-0 실크 봉합사와 신장 동맥과 정맥 아래의 대동맥을 Ligate. </li><li> 신장 hilum에서 방광 왼쪽 ureter을 해부하다. </li><li> 방광 돔에서 caudad 철회를 사용하여 ureterovesical 접합을 폭로. </li><li> 방광 - 투 - 방광 요도 문합를 통해 재건에 사용되는 왼쪽 ureterovesical 접합을 포함하는 방광의 엑사이스 작은 타원형의 패치. 오른쪽 ureter은 방광에 포함되어 있지 않습니다. </li><li> 8-0 실크 봉합사와 신장 동맥과 정맥 아래 IVC를 Ligate. </li><li> 신장 동맥과 정맥 위의 대동맥을 Ligate, 천천히 0.2 - 0.4ml 감기로 현장에서 그래프트를 perfuse, infrarenal 대동맥을 통해 링어의 락트 솔루션 heparinized. </li><li> 8-0 실크 봉합사와 신장 동맥과 정맥 위의 IVC를 Ligate. </li><li> IVC와의 교차점에서 신장 정맥을 가로로 쪼개다. </li><li> 신장 동맥 아래 약 2mm, obliquely 대동맥을 나눕니다. </li><li> 신장과 연방 EN 방광 패치에 연결된 ureter와의 혈관 공급을 제거합니다. 얼음 링어의 락트 산 용액에 보관하십시오. </li><li> 마취하에 자궁 경부 전위에 의해 기증 마우스를 안락사. </li></ol> 받는 사람 이식 (2) : <ol><li> 65 MG / kg pentobarbital IP를받는 사람을 마취와 Isoflurane으로 마취 (2-3 %)를 유지합니다. </li><li> , 정중선 절개를 확인 서부 유럽 표준시 거즈로 소장을 커버하고 조심스럽게 왼쪽으로 철회. </li><li> 오른쪽 ureter, 신장 동맥과 정맥을 Ligate, 오른쪽 신장을 제거합니다. </li><li> 조심스럽게 허리 지점을 ligating 이후 두 4 - mm microvasular 클램프와 infrarenal 대동맥과 IVC을 분리하고 교차 클램프. </li><li> 대동맥의 전체 두께 10-0 나일론 봉합사를 놓고 한 컷 (선박의 직경의 5 분의 1 정도)하여 타원형 aortaotomy를 만들기 위해 철회. </li><li> 30 게이지 바늘로 IVC를 관통하고 적절하게 microscissors를 사용하여 확장하여 길이 venotomy하십시오. </li><li> intraluminal 혈액이나 혈전을 취소 heparinized 호수와 대동맥과 IVC 모두 관개. </li><li> 얼음에서 기증자의 신장을 제거하고 마우스의 오른쪽 측면에 내부 abdominally 그것을 놓으십시오. 기증자 신장은 차가운 호수 - 절어 거즈 패드와 함께 촉촉한 보관됩니다. </li><li> 기증자의 대동맥을 재는 완과받는 사람의 aortaotomy의 근위 및 말초 꼭대기에 두 개 숙박 봉합합니다. </li><li> 대동맥의 외부에 2 ~ 3 연속 10-0 나일론 봉합을 사용하여 기증 대동맥 수갑을 채워받는 aortaotomy의 앞쪽에 벽을 사이의 엔드 - 투 - 사이드 문합를 확인합니다. </li><li> 받는 사람의 좌측으로 기증자 신장 이식을 돌립니다. 기증자 대동맥 수갑을 채워받는 aortaotomy의 후부 벽 사이의 이전 절차를 반복 </li><li> IVC의 내부에 4-5 지속 10-0 나일론 봉합을 사용하여 기증자 신장 정맥과받는 사람 IVC의 후부 벽 사이의 엔드 - 투 - 사이드 문합을 수행합니다. 그리고 IVC 및 기증자 신장 정맥의 앞쪽에 벽은 IVC 외부 4 ~ 5 연속 봉합과 함께 폐쇄됩니다. </li><li> 정맥과 동맥 모두의 문합 20 분 이내에 완료해야합니다. </li><li> 문합 동안 차가운 식염수로 여러 번 그래프트를 씻어. </li><li> 받는 사람 방광의 돔을 분할하고, 소작과 함께 출혈이 중지합니다. </li><li> 돔에 작은 절개를합니다. </li><li> 받는 사람의 방광 돔와 기증자 작은 방광 패치를 anastomize에 10-0 나일론의 두 숙박 봉합를 놓습니다. 문합의 각 측면은 8 ~ 10 연속 봉합사와 봉합합니다. </li><li> 연속 4-0 무균, 합성 흡수성 봉합사 vicryl 두 레이어의 복부를 닫습니다. </li><li> 간헐 주입 IV에 의해 수술 도중에받는 생쥐에게 0.4ml 따뜻한 호수주세요. </li></ol> 수신자의 포스트 수술 케어 <ol><li> 복부를 닫은 후, 페니실린 (500U/10g)의 하나 근육내 주사는 (2) 관리합니다. </li><li> 또한, 수신자가 고통을 하루 종일 0 1 일 게시 수술에 intramuscularly 0.05 밀리그램 / kg buprenorphine를 받게됩니다. 실험 테 때까지 수술 후, 이식 수신자 sulfatrim (100mg/kg)를 포함하는 물을받을 수rminated. </li><li> 첫 24 시간에 대한 postoperatively 생쥐는 배양기를 제어 온도에 보관하고 있습니다. </li><li> 마우스는 일반적으로 작업을 1 시간 이내에 마취에서 회복하고 정기적으로 음식과 물 광고 libitum 부여됩니다. </li><li> 피부 봉합 2 주 수술 후 제거됩니다. </li></ol> 수신자 신 <ol><li> 65 MG / kg pentobarbital IP를받는 4 ~ 7 일 포스트 신장 이식 마취, 마취는 Isoflurane (2-3 %)에 의해 유지됩니다. </li><li> 정중선 절개를 통해 복부를 엽니다. </li><li> 생리 - 젖었 거즈로 소장을 커버하고 신중하게 오른쪽으로 철회. </li><li> 왼쪽 ureter, 신장 정맥과 동맥을 ligating 후 왼쪽 신장을 제거합니다. </li><li> 연속 4-0 합성 흡수성 봉합사 vicryl 두 레이어의 복부를 닫습니다. </li></ol> 전체 혈액 크레아티닌 측정 <ol><li> 미만 0.2 CC의 격주 혈액 컬렉션 주간은 Isoflurane 마취 마취 또는 역 orbitally없이도 submandibularly 수행됩니다. </li><li> 양적 전체 혈액 크레아티닌 수치는 I - 합계 휴대용 임상 분석기 (Heska 공단, 포트 콜린스, CO)를 사용하여 결정됩니다. </li><li> 종래의 단위 (MG / DL)은 88.4로 종래의 단위를 곱하여 micromol / L의 SI 단위로 변환됩니다. </li></ol>

<ol>
	<li>Russell, P. S., Chase, C. M., Colvin, R. B., Plate, J. M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+transplants+in+mice.+An+analysis+of+the+immune+status+of+mice+bearing+long-term,+H-2+incompatible+transplants.">Kidney transplants in mice. An analysis of the immune status of mice bearing long-term, H-2 incompatible transplants.</a> JExpMed. 147, 1449-1468 (1978).</li><li>Zhang, Z., Schlachta, C., Duff, J., Stiller, C., Grant, D., Zhong, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+techniques+for+kidney+transplantation+in+mice.">Improved techniques for kidney transplantation in mice.</a> Microsurgery. 16 (2), 103-109 (1995).</li></ol>

동물 실험은 오하이오 주립 대학 IACUC에 의해 명시된 지침과 규정에 따라 수행되었다.

우리는&gt; 90 % 기술 성공률과 마우스에서 신장 이식의 약 1천2백가지 경우을 수행했습니다. 심장 이식에 비해 신장 이식의 성공률은 일반적으로 낮다. 그러나, 저희 연구실은 신장 이식의 높은 성공률을 (&gt; 90%) 유지하고 있습니다. 성공을위한 핵심 포인트는 기증자의 신장과받는 사람 모두에게 reperfusion의 부상을 감소하고, 절차 및 사후 operatively 전역 출혈 및 혈전증을 최소화하는 것입니다. 다?...

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<td>i-STAT</td>
<td>Other</td>
<td>HESKA</td>
<td>Cat 5101</td>
<td>&nbsp;</td>
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<td>Other</td>
<td>HESKA</td>
<td>CAT 5110-CREA</td>
<td>&nbsp;</td>
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<td>Sulfatrim</td>
<td>Other</td>
<td>Actavis</td>
<td>R02109</td>
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<td>Other</td>
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murine renal transplantation procedure

생쥐의 신장 이식 orthotopic은 기술적으로 도전적인 절차입니다. 생쥐에서 처음으로 신장 이식이 (1) 벌써 30 년 동안 러셀 외 수행하고 년후 장 외 (2)에 의해 정제했지만, 세계에서 몇 사람이 프로 시저를 마스터했습니다. 저희 연구실에서는 성공적으로&gt; 90 % 생존율과 1200 orthotopic 신장 transplantations을 수행했습니다. 성공을위한 핵심 포인트는 두 절차 및 사후 이식하는 동안 reperfusion 상해, 출혈 및 혈전증의 엄격한 통제를 포함하고, anastomoses 대신 11-0 봉합사의 10-0 중 하나를 사용하십시오. 받는 사람의 포스트 수술 관리 및 치료 성공과 평가를 이식하기 위해 매우 중요합니다. 모든 신장 이식의 수신자는 즉시 페니실린의 주사 지속적으로 마시는 물 속에 포스트 이식과 sulfatrim의 형태로 항생제를받을 수 있습니다. 전체 동물의 건강은 매일 전체 혈액 크레아티닌 분석이 이식편 기능을 평가하기위한 휴대용 I - STAT 기계와 함께 정기적으로 수행 평가됩니다.

Watch this Scientific Journal Video about Murine Renal Transplantation Procedure at JoVE.com

Murine Renal Transplantation Procedure

생쥐의 신장 이식은 성공을위한주의 이후 수술 진료와 치료를 필요로하는 기술적으로 도전적인 절차입니다.

Murine 신장 이식 절차

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Research

JoVE Journal

Biology

29.5K 조회수.  The Ohio State University. 생쥐의 신장 이식은 성공을위한주의 이후 수술 진료와 치료를 필요로하는 기술적으로 도전적인 절차입니다.

비디오: Murine Renal Transplantation Procedure

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

마우스의 Heterotopic 심장 이식

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

연구

생물학

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis.  In this article, we provide details of the technique to supplement the video.

The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

마우스에 작은 창자 이식

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

Murine Skin Transplantation

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take &lt;10 min to perform.

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c--&gt;C57BL/6 skin transplant.

Murine 피부 이식

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

인간 CD40 - 활성화된 B 세포의 생성

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

이식에 의해 키메라 Zebrafish의 배아를 생성

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

내유 상피 이식 절차

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Zebrafish의 Pronephros의 레이저 절제는 신장 상피 조직이 재생된를 공부

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

전체 세포 인구의 단일 셀 수준에서 Cyclin B의 Proteolysis 유학

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

기본 마우스 조골 세포에서 골 세포 배양을 구하는 레티노 산의 적용

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

유동 세포 계측법에 의한 신장 상피 세포에서 세포 내 칼슘 측정

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...