NIH 면역학 교육 그랜트 T32 AI 60,573 (KRG)와 NIH 부여 GM 41,514 (MDC)

이전 실험 개시하기 : <ul><li> IACUC 승인을 받아야 </li><li> 수술기구 및 Q - 도움말을 소독 </li></ul> I. 하베스트 기증자 조직 <ol><li> (기관의 SOP에 따라) 기증자 마우스를 안락사 </li><li> 알코올 (内耳)의 기지에서 젖은 머리 뒤로 </li><li> 기지에서 귀를 잘라 수확하여 기증자 피부 </li><li> 얼음 접시에 넣어 조직 </li><li> 분할 이어 : 쥐 - 치아 포셉과 (内耳)의 기지로 조직 핀치 복부와 지느러미 양측, 부드럽게 조직을 분열 시키려고, 부동 등의 조직, 내부 표면 아래, PBS 얼음 속에서, collagenous 복부 플랩를 버리고 </li></ol> II. 이식받는 준비 <ol><li> 마취 마우스 승인 절차를 사용 </li><li> 수술 통증 (예 : 0.05-0.1 MG / G Bupernex의 IP)에 대한 진통제 투여 </li><li> 가로 위치에 플레이스 마우스 </li><li> 동물 충분히 진정되도록 발끝 핀치를 수행 </li><li> 젖은 알코올로 머리와 흉부를 면도 </li><li> 알코올 면봉과 함께 깨끗한 지역을 닦아 </li></ol> III. 피부 이식 <ol><li> 쥐 - 치아 포셉와 함께 텐트 피부 포셉 아래 ~ 0.25-0.5 cm 잘라 : ~ 1cm 절개를 확인 </li><li> PBS에서 기증자 피부 이식 침대 위에 자리 (그래프트 침대에 직면하고 귀 피부의 내부 표면)를 제거하고, 멸균 면봉을 넣었와 여분의 PBS를 제거 </li><li> 그 이식 (이식 가장자리가 아래에 모습을 드러냅니다하지 그 이식받는 사람의 트렁크 피부에 숙취가없는 것을 확인하고) 이식 침대 이내에 단호하게 자리잡고 있으므로 신중하게 거리를 초과 기증자의 피부를 트림 </li><li> 멸균 붕대로 감싸받는 사람 (피부 이식을 통해 위치가 아닌 접착 거즈 세그먼트 포함) </li><li> 그 붕대이 (동물의 호흡을 평가) 너무 꽉되지 않도록하고 팔을 자유롭게 이동 아르 </li><li> 단일 2-0 실크 봉합사와 보안 붕대 </li><li> 깨끗한 새장에 플레이스 동물 (동물까지 가열 램프에 따라 충분히 마취로부터 복구되었습니다) </li><li> 모니터받는 고통의 흔적을 1-2/day과 고통 구호를위한 필요에 따라 진통제 투여 </li></ol> IV. 붕대 제거 (6~7일 포스트 이식) <ol><li> 마취 마우스 승인 절차를 사용 </li><li> 실크 봉합사 컷 및 제거 </li><li> 신중하게 붕대 (그래프트를 방해하지 않도록 만들기)를 통해 컷 뭉툭한 끝 가위를 사용하여 부드럽게 제거 </li><li> 깨끗한 케이지 마우스로 돌아가기 </li><li> 폼 또는 수축의 흔적을 붕대 제거 후 이식 ~ 8 시간을 확인합니다. 있는 경우, 수술이 vascularize에 실패 할 수도 있습니다 및 기술 실패로 간주되어야합니다. </li><li> 거절의 흔적 (보통 기증자의 조직 ~ 80 %의 괴사로 정의)에 대한 일일 모니터 </li><li> Allogeneic 피부 이식은 보통 D8 - 12 수술 사이 거부합니다 </li></ol> V. 유용한 힌트 <ol><li> 가까이 귀에의 기본에 기증자의 조직을 것입니다 수확 : 1) 그래프트의 크기를 극대화하고, 2) 분할을 용이하게합니다 고르지 복부 오버행을 제공합니다 </li><li> 항상받는 조작에 대한 새 면도날을 사용하여 </li><li> 진통이 IP를 부여하는 경우 (그래프트을 방해의 가능성을 최소화) 수술 처리를 최소화하기 위해 절차를 이식하기 전에 관리 </li><li> 가능하다면, 붕대를 적용하기 전에 장갑을 제거합니다. 장갑은 붕대를 심어 응용 프로그램을 복잡하게하는 경향이 </li><li> 붕대 끝에 하나의 봉합사를 게재하면 조기 제거를 최소화합니다 </li><li> 꽉로 허용하고 봉합을 배치하기 전에 호흡을 평가로 바꿈 붕대. 붕대가 너무 꽉 경우, 동물은 호흡을 중지합니다. 붕대가 너무 느슨한 경우, 동물은 그것을 제거됩니다. </li></ol>

The authors have nothing to disclose.

본 동영상 문서에 시연, 피부 이식 모델 allogeneic T 세포 응답을 모니터링을위한 빠르고 쉬운 방법입니다. Allogeneic 피부 이식은 어플 리케이션의 광범하고 분석하는 immunosuppressive 대리인 및 내성 유도 프로토콜뿐만 아니라 조사를 위해 다양한 차단 항체의 생물 학적 효과의 다양한의 효율성을 활용하고 있습니다.

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<td>Micro-dissecting forceps (curved)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F4142</td>
<td>&nbsp;</td>
<tr>
<td>Rat-tooth forceps (1x2 teeth, straight)</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11053-10</td>
<td>n=2</td>
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<td>Strabismus scissors (blunt end)</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14074-11</td>
<td>&nbsp;</td>
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<td>Iris forceps (curved)</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11065-07</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15006-09</td>
<td>&nbsp;</td>
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<td>Halsey micro needle holder</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>12500-12</td>
<td>&nbsp;</td>
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<td>10 cm plates</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430167</td>
<td>&nbsp;</td>
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<td>Cellgro</td>
<td>21-040-CV</td>
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<td>517201</td>
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<td>&nbsp;</td>
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<td>8881 600145</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Solon</td>
<td>362</td>
<td>&nbsp;</td>
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<td>Single edge razor blades (#9)</td>
<td>&nbsp;</td>
<td>Smith</td>
<td>67-0238</td>
<td>&nbsp;</td>
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<td>Silk suture (2-0; PS-2)</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>583H</td>
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murine skin transplantation

가장 엄격한 최소 기술적으로 도전적인 모델 중 하나로서, 피부 이식은 MHC - 서로 다른 기증자 항원을 분석 호스트 T 세포 응답에 대한 표준 방법입니다. 이 비디오 문서의 목적은 마우스 모델을 사용 피부 이식의 단계별 영상 데모로 뷰어를 제공하는 것입니다. 2) 이식에 대한 수신자를 준비, 3) 피부 이식, 4) 붕대 제거 및 모니터링 이식 거부, 5) 유용한 힌트 1) 수확 기증자 피부 : 프로토콜은 5 주요 구성 요소로 나누어져 있습니다. 일단 실력, 절차 자체가 수행하는 &lt;10 분 정도 소요됩니다.

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

Murine Skin Transplantation

Allogeneic 피부 이식은 MHC - 서로 다른 기증자 항원을 분석 호스트 T 세포 응답에 대한 표준 모델입니다. 이 비디오 문서 BALB/c--&gt; C57BL / 6 피부 이식을 수행에 관련된 각 단계의 영상 자습서를 제공합니다.

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 ...

Research

JoVE Journal

Biology

22.4K 조회수.  University of California, Irvine (UCI). Allogeneic 피부 이식은 MHC - 서로 다른 기증자 항원을 분석 호스트 T 세포 응답에 대한 표준 모델입니다. 이 비디오 문서 BALB/c--> C57BL / 6 피부 이식을 수행에 관련된 각 단계의 영상 자습서를 제공합니다.

비디오: Murine Skin Transplantation

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 Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

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 ...

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 ...

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal2. The use of donor males carrying the bacterial &beta;-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals1. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located3. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation4,5, to study Sertoli cell-germ cell interaction6,7, SSC homing and colonization3,8, as well as SSC self-renewal and differentiation9,10.
Germ cell transplantation is also feasible in large species11. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species12. An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm13. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice14.

Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.

배아 세포 이식 및 Testis 조직 마우스에 Xenografting

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

Planarian의 부동화, 부분 조사 및 조직 이식

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

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 ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

근육 위성 세포의 분리, 문화, 및 이식

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

An Immunofluorescent Method for Characterization of Barrett&#8217;s Esophagus Cells

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One of the primary risk factors of EAC is Barrett&#8217;s esophagus (BE), a metaplastic change of the normal squamous esophagus in response to chronic heartburn. Despite the well-established connection between EAC and BE, interrogation of the molecular events, particularly altered signaling pathways involving progression of BE to EAC, are poorly understood. Much of this is due to the lack of suitable in vitro models available to study these diseases. Recently, immortalized BE cell lines have become commercially available allowing for in vitro studies of BE. Here, we present a method for immunofluorescent staining of immortalized BE cell lines, allowing in vitro characterization of cell signaling and structure after exposure to therapeutic compounds. Application of these techniques will help develop insight into the mechanisms involved in BE to EAC progression and provide potential avenues for treatment and prevention of EAC.

There is a discernible need for improved information on molecular drivers of Barrett&#8217;s Esophagus. Immunofluorescent staining is a useful technique for understanding the effects of cell signaling on cell morphology. We present a simple, effective protocol for the use of immunofluorescent staining to assess therapeutic treatment in Barrett&#8217;s Esophagus cells.

바렛 식도 세포의 특성에 대한 면역 방법

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One ...