만들기 유전자 총 총알 (골드) <ol><li> 100 % EtOH에서 100X PVP (0.5mg/ml)를 확인합니다. </li><li> BioRad 1.6 마이크론 microcarrier 골드의 30mg을 무게와 1.5 ML eppendorf 튜브에 넣습니다. </li><li> 0.5-3 μg 플라스미드 / MG 황금의 농도를 제공하는 데 필요한 플라스미드의 양을 계산하고, 황금 microcarriers와 튜브에 플라스미드를 놓습니다. 필요한 경우 여러 plasmids을 결합합니다. </li><li> 증류수 100 μl에 플라스미드의 볼륨을 가져와. </li><li> 튜브에 0.05M spermidine 100 μl를 추가합니다. 소용돌이 관 30 초전. </li><li> 와동 전속력으로 일분. 3~5분에 대한 Sonicate .. </li><li> 15 분 동안 Biorad의 PDS-1000/He 장치에 Tefzel 튜브의 적절한 길이를 건조시킵니다. </li><li> 전속력으로 1~2분에 대한 sonication 후, 소용돌이. vortexing하면서 튜브를 열 수 있도록 천천히 속도를 줄입니다. </li><li> vortexing하면서 현명한 튜브 드롭 1 M CaCl 천천히이 100 μl를 추가합니다. </li><li> 10 분 실온에서 튜브를 남겨주세요. </li><li> 5 미크론 필터를 확인하십시오. <ol><li> 바늘없이 2 5ml 주사기를 가져가라. </li><li> 플런저를 제거하고, 검은 모자를 분리합니다. 작은 가위 복용, 반지를 줄 수있는 블랙 캡의 일부를 잘라. NET이 고리에 다른 플런저를 반복합니다. </li><li> 샌드위치 5 마이크론 (소형 부품 (주) 나일론 필터, ½ 인치 직경) 두 고리 사이의 필터와 주사기의 끝부분에 샌드위치를​​ 누르십시오. </li><li> 50ml 원뿔 플라스크 위에 매달려 주사기. </li></ol></li><li> 십분 후, 와동 30 초 튜브. </li><li> 30 초 동안 튜브를 원심하여 펠렛을 저장합니다. </li><li> 펠렛에 100 % EtOH의 1ml을 추가합니다. 펠렛, 그리고 소용돌이 30 초 헤어지고 피펫 팁을 사용합니다. 30 초 돌려서 EtOH를 제거합니다. 두 번 더 반복합니다. </li><li> (끝 헤어) 펠렛에 100 % EtOH의 1ml을 추가합니다. 5 마이크론 필터로 이것을 놓고 50ml 원뿔 튜브로 중력에 의해 흐름을 통해 그것을하자. 튜브를 세척하거나 통해 흐름에 도움이 필요한 경우 더 많은 EtOH를 사용합니다. </li><li> 50~10분 위해 원심 분리기에서 2000rpm에서 50ml 튜브를 스핀. 뜨는 저장, 펠렛을 제거합니다. </li><li> 800μl - 1.5ml 백퍼센트 EtOH 및 해당 8 μl - 15 μl PVP 솔루션을 추가 필요 microcarriers의 원하는 밀도에 따라 다릅니다. </li><li> 소용돌이 즉시 Biorad의 PDS-1000/He 장치로 이동합니다. </li><li> Biorad PDS-1000/He 장치 <ol><li> 건조 튜브에 N2 가스 탱크를 연결하는 튜브를 분리 </li><li> 컴퓨터에서 Tefzel 튜빙을 건조 꺼내서 </li><li> 50ml 원뿔 관에 한쪽 끝을 삽입하고 튜브의 다른 끝에 주사기를 연결합니다. 튜브의 중간에 갈색 슬러리를 빨아. </li><li> 기계로 튜브를 다시 삽입합니다. </li><li> 4 분간 (회전없이) 잠깐 만요 </li><li> 사분 후 microcarriers 방해하지 않도록하고, 천천히 그리고 신중하게 EtOH를 빨아. </li><li> 1 분 튜빙을 회전합니다. </li><li> 일분 후, 건조 튜브에 N 2 가스 탱크를 연결하는 튜브를 연결하고 15 분 동안 튜브가 건조하게. </li></ol></li><li> 십오분 후, exsiccator 4 ° C 즉시 사용되거나 저장될 총알 크기​​에 튜브를 잘라. 총알 3 개월까지 저장할 수 있습니다. </li></ol> 유전자 군의 준비 <ol><li> 카트리지에 플레이스 총알. 망막의 각 조각은 1-3 총알을 맞았 수 있습니다. </li><li> 유전자 군에 카트리지를 삽입하고 헬륨 소스에 총을 부착합니다. 압력 110 PSI 회원으로 설정합니다. </li></ol> 미디어 준비 <ol><li> 해부 매체 <table style="width: 229px; height: 157px;" border="0"><tbody><tr><td> 1 병 에임스 솔루션 (시그마, 8.8g) </td></tr><tr><td> 1.9 GM의 중탄산 </td></tr><tr><td> 10 ML 1 % 펜 / strep / L - 글루타민 (1:100) (Gibco / Invitrogen) </td></tr><tr><td> 990 ML 증류수 1L에 </td></tr><tr><td> ------------------------------- </td></tr><tr><td> 1 리터 </td></tr></tbody></table> </li><li> 0.22 μm의 필터를 사용하여 절개 매체를 필터링합니다. </li><li> 배양 매질 (500ml) <table style="width: 283px; height: 157px;" border="0"><tbody><tr><td> 필터링 해부 미디어의 485 ML </td></tr><tr><td> 1 % N2의 보충 5 ML (Gibco / Invitrogen) </td></tr><tr><td> 10ml 1퍼센트 말 혈청 (시그마) </td></tr><tr><td> 페놀 레드 500 μl </td></tr><tr><td> -------------------------------------------------- - </td></tr><tr><td> 0.5 L </td></tr></tbody></table> </li><li> 0.22 μm의 필터를 사용하여 배양 매체를 필터링합니다. </li><li> 망막의 수확 절차를 시작하기 전에 15 분 해부 매체에 거품 O 2. </li></ol> 망막에 대한 배양 챔버의 준비 <ol><li> 후드에서 배양 매질의 25 ML 여러 60mm 깊이 요리 (인큐베이션 회의소, Nunc)를 입력합니다. 요리의 수는 RET의 숫자에 따라 달라집니다inas이 필요 해요. </li><li> 각 깊은 그릇에 필터 스탠드를 놓습니다. 스탠드만이 매우 팁이 침수되지 않습니다. </li><li> 망막 준비가 될 때까지 인큐베이터에 모두 깊은 요리를 놓습니다. </li><li> 이 100mm 배양 접시를 기입하고 해부 벤치에 가져와. </li></ol> 토끼 망막의 준비 <ol><li> 설립 프로토콜에 따라 토끼를 희생. 다음은 후드 아래에 수행할 수 필요가 없습니다. </li><li> 직각 가위로 눈을에 연결된 안정 및 광학 신경을 절단, 토끼의 눈 소켓에 눈 뒤에 그것을 넣습니다. </li><li> 부드럽게 눈을 밖으로 리프트 및 산소 해부 매체로 씻으십시오. </li><li> 눈 슬라이의 우물에 눈을 (각막가 직면한) 장소와 뚜껑을 닫습니다. </li><li> 수평으로 바로 뚜껑 아래에있는 블레이드를 놓습니다. 단칼에에서 눈을 컵을 떠나, 각막을 차단하기 위해 눈을 통해 칼날을 그립니다. </li><li> 포셉를 사용하여, 잘 눈 슬라이에서 망막을 제거하고 필터 종이에 그것을 놓으십시오. </li><li> 절단 시신경 근처 지역을 클램핑하고 필터 종이에 뒤로 당겨 유리와 렌즈를 제거합니다. 거의 모든 유리가 제거되고 아이 컵이 deflated 보이는 때까지 여러 번 반복합니다. </li><li> 씻어 해부 미디어와 페트리 접시에 눈 컵 플레이스. </li><li> 조각의 원하는 번호 (5 개 최대)로 눈을 컵 컷. </li><li> 아이 컵 한 조각을 가지고 현미경으로 그것을 놓으십시오. </li><li> 포셉를 사용하여 망막의 가장자리에 가볍게 함께 공막엔 및 맥락막 고정. 다른 한편으로 바로 맥락막과 망막 사이에 브러시에 좋은 브러시를 사용합니다. </li><li> 그것이 분리되는 때까지 짧은 부드러운 동작 사용하여 맥락막에서 망막을 괴롭혀. </li><li> 멸균 피펫 전송에서 1 인치 컷. 씻어 배양 매체의 페트리 접시에 망막과 장소를 빨아 이것을 사용하십시오. </li><li> 1 인치 다른 살균 전송 피펫을 사용하면 0.4 μm의 Millicell 필터 (PICMORG50 Millipore, 고양이도없는)에 망막을 전송 주의깊게, 잘라. </li><li> 위쪽을 향하고 신경절 세포 면을 방향 망막에 브러시를 사용합니다. </li><li> 망막의 가장자리에 가볍게 빗질하여 망막을 평평하게. 최대한 브러시와 신경절 세포 레이어를 감동 최소화합니다. </li><li> 이전 피펫과 필터 초과 매체를 제거합니다. </li><li> 포셉 사용하여 수정된 suctioner에 필터를 전송합니다. suctioner에있는 필터의 모든 배양 매질을 제거하려면 2-3 시간을 그립니다. </li></ol> 유전자 거닝와 망막의 인큐베이션 <ol><li> 후드 아래, 60mm 깊이 요리 (배양 챔버)에 필터에 서 망막 함유 - 필터의 각 장소. </li><li> 매체는 필터의 아래쪽에 접촉하고 매체가 망막에 필터의 측면을 통해 유출하지 않는 것을 확인하십시오. 필터는 망막과 매체 사이의 인터페이스 역할을한다. </li><li> 총알이 두 발과 망막의 각 조각을 쏴. 바로 조직 위에 플레이스 유전자 총을에 대한 3~4센티미터 거리. </li><li> 37 ° C 배양기 - 35 흔드는에 모든 배양 챔버를 놓습니다. </li><li> 배양 매질 매일 교체하십시오. 망막은 6 일 최대 incubated 수 있습니다. </li></ol>

<ol>
	<li>Landreth, . B. W., Agranoff, N. R., Smalheiser, S. M., Crain, M. B., Bornstein, R., Adler, P. J., Magistretti, A. G., Hyndman, W. J., Shoemaker, H., Rehm, T., Schafer, H., Betz, S. U., Kim, H., Takahashi, A. R., Caffe, A., Soderpalm, T., van Veen, H. C., Wong, S., Thompson, D. Y., Yu, S. J., Cringle, V. A., Alder, S. J., Taylor, G. M., Seigel, A. R., Caffe, P., Ahuja, B., Holmqvist, S., Azadi, J., Forsell, I., Holmqvist, J., Hatakeyama, R., Kageyama, S. S., Zhang, X. Y., Fu, C. J., Barnstable, S. W., Wang, X., Mu, W. J., Bowers, W. H., Klein, T., Inoue, M., Hojo, Y., Bessho, Y., Tano, J., Lee, R., Kageyama, A., Kretz, S. H., Hermening , S., Isenmann , I., Liljekvist-Larsson, K., Johansson, B., Reidel, W., Orisme, T., Goldmann, W. C., Smith, U., Wolfrum, H., Xin, J. A., Yannazzo, R. S., Duncan, E. V., Gregg, M., Singh, P., Koulen, S. A., Johnston, D. C., Tang, D. C., Lo, A. K., McAllister, K. a. t. z., LC, L. E., Rajagopalan, J. S., Malter, A. K., McAllister, W. -. B., Gan, J., Grutzendler, W., Wong, R., Wong, J., Lichtman, H., Sato, S., Hattori, S., Kawamoto, I., Kudoh, A., Hayashi, I., Yamamoto, G., Zhang, M. E., Selzer, R. L., Sohn, M. T., Murray, K., Schwarz, J., Nyitray, P., Purray, A. P., Franko, J. A., O'Brien, M., Holt, G., Whiteside, S. C., Lummis, M. H., Hastings, L., Klimaschewski, W., Nindl, M., Pimpl, P., Waltinger, K., Pfaller, P., Kettunen, J., Demas, C., Lohmann, N., Kasthuri, Y., Gong, R. O., Wong, N., Gamper, M. S., Shapiro, M. J., Wirth, P., Wahle, J., O'Brien, S. C., Lummis, A. M., Benediktsson, S. J., Schachtele, S. H., Green, M. E., Dailey, L., Chow, E. M., Levine, T. A., Reh, R. L., Rockhill, F. J., Daly, M. A., MacNeil, S. P., Brown, R. H., Masland, W., Sun, N., Li, S., He, W., Sun, N., Li, S., He, R. C., Stacy, R. O., Wong, E., Strettoi, V., Pignatelli, C., Rossi, V., Porciatti, B., Falsini, V., Pignatelli, E., Strettoi, V. P., Connaughton, D., Graham, R., Nelson, P. H., Edqvist, F., Hallbook, T. C., Jakobs, R. T., Libby, Y., Ben, S. W., John, R. H., Masland, Y., Qin, G., Xu, W., Wang, R., Roizenblatt, J. D., Weiland, S., Carcieri, G., Qiu, M., Behrend, M. S., Humayun, A., Koizumi, G., Zeck, Y., Ben, R. H., Masland, T. C., Jakobs, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explant+culture+of+adult+goldfish+retina:+a+model+for+the+study+of+CNS+regeneration.">Explant culture of adult goldfish retina: a model for the study of CNS regeneration.</a> Brain Res. 161, 39-55 (1979).</li></ol>

1. 이 방법으로 어떤 작업을 할 수 있습니까? <ul><li> 명확하게 세포 형태를 참조하십시오. </li><li> 그들의 기록 라벨 세포. </li><li> Overexpress의 PSD95 같은 단백질,뿐만 아니라 세포의 반응을 조절 다른 단백질, 예를 들어 이온 채널, 수용체. </li><li> 억제 RNAS을 표현한다. </li></ul> 2. 얼마나 오래 성인 망막을 보호할 수 있습니까? <ul><li> 사일는 성인 토끼에 대한 문제가 없습니다. 육일 후 세포가 &quot;아픈&quot;봐,...

organotypic culture of adult rabbit retina

기능 신경 조직의 Organotypic 문화 시스템은 신경 생물학 연구에 중요한 도구입니다. 이상적으로, 이러한 시스템은 이미징 기술, 유전자 조작, 그리고 electrophysiological 녹화와 호환되어야합니다. 여기는 전문 장비와 거의 유지 보수가 필요없는 성인 토끼 망막에 대한 간단한 계면 조직 문화 시스템을 제시한다. 우리는 토끼 망막과 망막 신경절 GFP 또는 세포에 subcellular 마커의 다양한 인코딩 plasmids의 입자 - 중재 유전자 전송의 해부와 부화를 보여줍니다. 토끼 망막이 방법은 최대 거의 전체 해부 구조의 변경이나 개별의 형태와 신경절 및 amacrine 세포, 6 일 동안 살아 있어야만 수 있습니다 교양.

Watch this Scientific Journal Video about Organotypic Culture of Adult Rabbit Retina at JoVE.com

Organotypic Culture of Adult Rabbit Retina

이 문서는 토끼 망막과 망막 신경절 GFP 또는 세포에 subcellular 마커의 다양한 인코딩 plasmids의 입자 - 중재 유전자 전송의 해부와 인큐베이션을 보여줍니다.

어른 토끼 망막의 Organotypic 문화

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

organotypic-culture-of-adult-rabbit-retina

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JoVE Journal

Biology

11.9K 조회수.  MGH - Massachusetts General Hospital. 이 문서는 토끼 망막과 망막 신경절 GFP 또는 세포에 subcellular 마커의 다양한 인코딩 plasmids의 입자 - 중재 유전자 전송의 해부와 인큐베이션을 보여줍니다.

비디오: Organotypic Culture of Adult Rabbit Retina

Horizontal Slice Preparation of the Retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of retinal neurons, such as amacrine cells, expand their dendrites horizontally, so that the morphology of the cells is supposed to be severely damaged in the vertical slices. In the whole-mount preparation, especially for patch-clamp recordings, retinal neurons in the middle layer are not easily accessible due to the extensive coverage of glial cell (Mueller cell) s endfeets. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact. The slice was made horizontally at the inner layer of the retina using a vibratome slicer after the retina was embedded in the low-temperature melting agarose gel. In this horizontal slice preparation of the retina, we studied the function of retinal neurons compared with their morphology, by using patch-clamp recording, calcium imaging technique, immunocytochemistry, and single-cell RT-PCR.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.

망막의 수평 슬라이스 준비

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

연구

생물학

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

E18 랫 두뇌의 Organotypic 슬라이스 문화

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

성인 쥐 심장 Myocytes의 차 문화

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

에서 Organotypic Murine 망막 Wholemount 문화 생체내 같은

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

성인 및 배아 골격근의 Microexplant 문화 및 골격 근육 줄기 세포의 분리

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

사람의 피부에서 성인 상피 줄기 세포의 분리 및 문화

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Morpholinos의 생체내의 Electroporation에서 성인 Zebrafish의 망막에

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

성인 골격근의 개인 Myofibers 및 위성 세포의 분리와 문화

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

절연, 문화, 그리고 성인 마우스 된 심근의 기능적 특성

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

성인 마우스 앞니의 치아 상피 줄기 세포의 분리 및 문화

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...