Estes estudos foram suportados em parte pelo financiamento pelo NIH R01 AI36532 para GAH. Gostaríamos de agradecer o apoio de destaque no transplante de patologia fornecidos pelo Dr. Tibor Nadasdy, ea orientação de valor inestimável e previsão fornecida pelo falecido Dr. Charles G. Orosz em iniciar este esforço de pesquisa.

Colheita de doadores de órgãos (2): <ol><li> Anestesiar o doador utilizando 65mg/kg IP pentobarbital; anestesia é mantida por isoflurano (2-3%). </li><li> Faça uma incisão na linha média do esterno às pubis para entrar no abdômen do doador e expor o rim esquerdo, deslocando o intestino lateralmente para o lado direito. </li><li> Isolar o rim esquerdo por ligadura e dividindo os vasos adrenal e testicular com um fio de seda 8-0. </li><li> Mobilizar a aorta acima de sua junção com a artéria e veia renal. </li><li> Mobilizar a veia cava inferior (VCI) acima e abaixo de sua junção com a artéria e veia renal. </li><li> Ligadura da aorta abaixo da artéria renal e veia com um fio de seda 8-0 para minimizar o sangramento. </li><li> Dissecar o ureter esquerdo do hilo renal até a bexiga. </li><li> Expor a junção ureterovesical usando retração caudal na cúpula da bexiga. </li><li> Especiais de consumo um patch, pequena elíptica da bexiga contendo a junção ureterovesical esquerdo, utilizado para a reconstrução urinária através de uma anastomose da bexiga a bexiga. O ureter direito não está incluído com a bexiga. </li><li> Ligadura da VCI abaixo da artéria renal e veia com um fio de seda 8-0. </li><li> Ligadura da aorta acima da artéria renal e veia e, lentamente, perfundir o enxerto in situ com 0,4 ml de 0,2 frio, heparinizado solução de Ringer lactato através da aorta infra-renal. </li><li> Ligadura da VCI acima da artéria renal e veia com um fio de seda 8-0. </li><li> Transecto da veia renal na sua junção com o IVC. </li><li> Divida a aorta obliquamente, cerca de 2 milímetros abaixo da artéria renal. </li><li> Remover o rim e sua fonte vascular com ureter anexado ao remendo de bexiga em bloco. Loja em solução Ringer com lactato no gelo. </li><li> Euthanize o mouse doador por deslocamento cervical sob anestesia. </li></ol> Transplante de destinatário (2): <ol><li> Anestesiar o destinatário IP com 65 mg / kg pentobarbital e manter a anestesia com isoflurano (2-3%). </li><li> Faça uma incisão na linha média, cobrir o intestino com uma gaze molhada e com cuidado retirar-la para o lado esquerdo. </li><li> Ligadura do ureter direito, artéria e veia renal, e remover o rim direito. </li><li> Cuidadosamente isolar e cross-clamp da aorta infra-renal e veia cava inferior com duas pinças de 4 mm microvasular após a ligadura dos ramos lombares. </li><li> Coloque um fio de náilon 10-0 através de uma espessura total de aorta e retrair para fazer uma aortaotomy elíptica por um único corte (cerca de um quinto do diâmetro do vaso). </li><li> Faça uma venotomia longitudinal perfurando a veia cava inferior com uma agulha de calibre 30 e estender adequadamente com microtesoura. </li><li> Irrigar tanto a aorta e veia cava inferior com solução salina heparinizada para limpar o sangue intraluminal ou coágulos. </li><li> Remover o rim de doador do gelo e coloque-o intra-abdominal no flanco direito do mouse. O rim do doador é mantida úmida com uma gaze embebida com solução salina fria. </li><li> Coloque duas suturas permanecer no ápice proximal e distal do aortaotomy destinatários com a braçadeira aórtica doadores. </li><li> Faça uma anastomose término-lateral entre a braçadeira aórtica doador ea parede anterior do aortaotomy destinatários, utilizando 2 ~ 3 contínua 10-0 suturas de nylon na parte externa da aorta. </li><li> Vire o enxerto renal de doador até o flanco esquerdo do destinatário. repita o procedimento anterior entre a braçadeira aórtica doador ea parede posterior da aortaotomy destinatários </li><li> Executar uma anastomose término-lateral entre a veia do doador renal e da parede posterior da VCI destinatários, utilizando 4-5 contínua 10-0 suturas de nylon no interior da VCI. e, em seguida, a parede anterior do IVC e doadores veia renal são fechadas com 4 ~ 5 sutura contínua fora da VCI. </li><li> Anastomose da veia e artéria ambos deve ser concluído dentro de 20 minutos. </li><li> Lavar o enxerto com solução salina fria várias vezes durante a anastomose. </li><li> Divide a cúpula da bexiga destinatário, e parar o sangramento com cauterização. </li><li> Faça um pequeno corte na cúpula. </li><li> Coloque duas suturas estadia de nylon 10-0 para os doadores anastomize bexiga pequena mancha com a cúpula da bexiga destinatários. Cada lado da anastomose é suturada com 8 ~ 10 pontos contínuos. </li><li> Fechar o abdômen em duas camadas com 4-0 estéril, fio absorvível sintético vicryl. </li><li> Dê ratos destinatário salina morna 0,4 ml durante a cirurgia por injeção intermitente IV. </li></ol> Cuidados pós-operatórios de destinatário <ol><li> Após o fechamento do abdome, uma única injeção intramuscular de penicilina (500U/10g) é administrado (2). </li><li> Além disso, os destinatários recebem buprenorfina 0,05 mg / kg por via intramuscular no dia 0 e cirurgia dia pós 1 para dor. Após a cirurgia, pacientes transplantados recebem água contendo sulfatrim (100mg/kg) até que o experimento é terminated. </li><li> Para primeiras 24h pós-operatório os ratos são mantidos em uma temperatura controlada incubadora. </li><li> Camundongos geralmente se recupera da anestesia dentro de 1 hora da operação e recebem comida regular e água ad libitum. </li><li> Suturas são removidas duas semanas após a cirurgia. </li></ol> Destinatário nefrectomia <ol><li> Anestesiar o destinatário 4 ~ 7 dias pós-transplante renal com 65 mg / kg IP pentobarbital; anestesia é mantida por isoflurano (2-3%). </li><li> Abrir o abdómen através de uma incisão mediana. </li><li> Cobrir o intestino com solução salina, gaze e com cuidado retirar-la para o lado direito. </li><li> Remover o rim esquerdo após ligadura à esquerda ureter veia renal e artéria. </li><li> Fechar o abdômen em duas camadas com fio absorvível 4-0 vicryl sintéticos. </li></ol> As determinações de creatinina Sangue Total <ol><li> Semanais para coleta de sangue a cada duas semanas, inferior a 0,2 cc é realizada submandibularly sem anestesia ou retro-orbital sob anestesia isoflurano. </li><li> Quantitativas dos níveis de creatinina no sangue são determinados utilizando um I-Stat Analyzer portátil Clínica (Heska Corp, Fort Collins, CO). </li><li> Unidades convencionais (mg / dl) são convertidos em unidades SI de micromol / L multiplicando as unidades convencionais por 88,4. </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>

Experimentos em animais foram realizados de acordo com as diretrizes e regulamentos estabelecidos pela The Ohio State University IACUC.

Efetuamos cerca de 1200 casos de transplante renal em ratos com uma taxa de sucesso técnico de&gt; 90%. Comparado com o transplante cardíaco, a taxa de sucesso de transplante renal é geralmente baixa. No entanto, nosso laboratório tem mantido uma alta taxa de sucesso (&gt; 90%) de transplante renal. Os pontos-chave para o sucesso são para reduzir as lesões de reperfusão a ambos os rins de doadores e receptores, bem como minimizar o sangramento e trombose durante todo o procedimento e pós-operatório. Os seguinte...

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<td>i-STAT</td>
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<td>Other</td>
<td>HESKA</td>
<td>CAT 5110-CREA</td>
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<td>Other</td>
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murine renal transplantation procedure

Transplante renal ortotópico em ratos é um procedimento tecnicamente desafiador. Embora o primeiro transplante renal em ratos foram realizadas por Russell et al mais de 30 anos (1) e refinada por Zhang et al anos depois (2), poucas pessoas no mundo têm dominado este procedimento. Em nosso laboratório temos realizado com sucesso 1.200 transplantes renais com ortotópico&gt; taxa de sobrevivência de 90%. Os pontos-chave para o sucesso incluem o controle rigoroso do sangramento reperfusão, e trombose, tanto durante o procedimento e pós-transplante, e uso de 10-0, em vez de 11-0 sutura para anastomoses. Pós-operatório cuidados e tratamento do destinatário é extremamente importante para o sucesso do transplante e da avaliação. Todos os destinatários do enxerto renal receber antibióticos sob a forma de uma injeção de penicilina imediatamente pós-transplante e sulfatrim na água de beber continuamente. Saúde animal global é avaliado diariamente e análises de creatinina no sangue são realizados rotineiramente com uma máquina portátil I-STAT para avaliar a função do enxerto.

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

Murine Renal Transplantation Procedure

Transplante renal em ratos é um procedimento tecnicamente desafiador que requer cuidados pós-operatórios cuidado e tratamento para o sucesso.

Murino Procedimento de Transplante Renal

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 visualizações.  The Ohio State University. Transplante renal em ratos é um procedimento tecnicamente desafiador que requer cuidados pós-operatórios cuidado e tratamento para o sucesso.

Vídeo: 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.

Transplante cardíaco heterotópico em ratos

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

Biologia

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.

Transplante de intestino delgado em ratos

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.

Transplante de pele murinos

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.

Geração de humanos CD40-activated células B

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.

A criação de embriões quiméricos Zebrafish por Transplante

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.

Procedimento de transplante do epitélio mamário

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.

Ablação a laser da Pronephros Zebrafish para estudo da regeneração epitelial renal

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.

Estudar proteólise de ciclina B no nível da célula individual em populações de células inteiras

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;

A aplicação de ácido retinóico para obter culturas de osteócitos principal do mouse osteoblastos

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.

Medidas cálcio citosólico em células epiteliais renais por citometria de fluxo

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