이 작품은 DKOD에 NIH 부여 NS27501에 의해 지원되었다. 이 작품에 대한 추가 지원은 HHMI 교수 프로그램을 통해 DKOD 지원하는 UC 어바인에 부여에 의해 제공되었다.

culturing의 하루 전에 준비 : <ol><li> 멸균 해부 솔루션을 확인하십시오. </li><li> 멸균 DMEM 만들기 4 10 ML aliquots에 보관 ° C를 2 주 동안. </li><li> 1 개월 50 또는 100 μl aliquots에 살균 DDM2 보충 및 동결하십시오. </li><li> 코나 / laminin하십시오. </li><li> 외투 coverslips. 옵션 : 최대 4 개월까지 냉동 살균 CNBM하고 저장하십시오. </li></ol> culturing의 날에 나도 층류 흐름 후드의 효소 솔루션 (ES)를 준비 <ol><li> 15 ML의 원심 분리기 튜브의 솔루션 (DS)를 해부 5 ML 놔. </li><li> 0.6 ML eppendorf에 L - 시스테인의 0.8 MG를 저울질하고 DS 150 μl를 추가합니다. 결정들이 갈 때까지 최대 피펫 아래로. </li><li> DS / 5 ML의 DS에 시스테인 150 ML을 추가하고 잘 섞는다. </li><li> Papain 50 U을 (부대) 추가합니다. </li><li> 0.1 N 나 OH 7 ML을 추가하고 잘 섞는다. Solution은 30 분 이내에 삭제됩니다. 활성화되면. </li><li> 멸균 1.5 ML 스크류 캡 microfuge 관에 0.2 mm의 주사기 필터와 필터 효소 솔루션 Papain : 5 ML의 DS 50 U를 원하십니까. 각 배치는 약간의 차이이므로, 얼마나 계산해야합니다 : 37.3 MG / ML 및 28.3 U / MG 37.3 X 28.3 = 1055.59 U / ML (1000 ML) 50 U = 47.4 ML </li></ol> II. DDM2 미디어 만들기 culturing의 당일 : DDM2를 만들기 위해 다음과 같은 보충제를 추가 DMEM의 10 ML은 culturing 전에 보충제를 추가합니다 : <ol><li> 100μl 트랜스페린 </li><li> 100μl Putrescine </li><li> 100μl 셀레늄 </li><li> 100μl Progesterone </li><li> 50μl 인슐린 </li><li> 10μl 20 Hydroxyecdysone </li></ol> 참고 : 계획 모두 문화 (각 두뇌 개인 35mm 페트리 접시에 하나 coverslip, 1.5 미디어 MS / 문화 도금)에 대한 충분한 DDM2 확인 단계 III - VI는 한 nonsterile 실험실 벤치에있을 수있는 45 분 이내에 완료해야합니다. III. Pupae 콜렉션 <ol><li> 해부 현미경 유리병의 측면 10-15 pupae을 선택합니다. </li><li> 마른 35mm 페트리 접시의 뚜껑에 플레이스 pupae. </li></ol> 참고 : 우리가 일반적으로 번데기 형성 (APF) 이후 55-78시간 사이 pupae의 두뇌를 사용합니다. neurite 가지의 전반적인 수준이 가장 오래된 pupae에서 약간 낮은 상태에서 머리 캡슐이 붕괴하는 경향 때문에 이것은 젊은 pupae에서 두뇌를 해부하다하는 약간 어렵습니다. 캔턴 - S wildtype 스트레인에서 다음 단계가 다른 작은 안료와 약간 붉은 갈색 빛을 아르 색소 눈이 인정하고 있습니다. IV. 해부 현미경으로 잘린 <ol><li> 플레이스 다음 페트리 접시의 뚜껑에 살균 해부 솔루션의 두 방울은 pupae 수 있습니다. </li><li> 부드럽게 왼손에 좋은 포셉로 단일 번데기를 개최하고 번데기의 앞쪽에 끝에있는 표피를 제거하려면 오른쪽에있는 1 CC의 주사기에 부착된 27 게이지 주사 바늘을 사용합니다. </li><li> 포셉와 약간 번데기를 당기 머리가 나온다으로 목을 자르다 27 게이지 바늘을 사용합니다. </li><li> 바늘이나 집게의 끝부분으로 솔루션을 해부의 드롭에 머리를 전송할 수 있습니다. </li><li> 당신은 한 방울의 10-15 고개를 때까지 반복합니다. </li></ol> 해부 현미경으로 머리에서 두뇌의 V. 제거 <ol><li> 각 손에 27 게이지 바늘로 1 CC의 주사기를 개최. </li><li> 코 당신과 머리 등의 표면 북쪽에 직면 있도록 솔루션을 해부의 드롭에있는 파리 머리의 위치를​​ 다음을 사용하십시오. </li><li> 머리를 아래로 핀과 등의 표면에 복부에서가는 오른쪽 눈에 슬릿을 수있는 권리 바늘을 사용하는 코에 왼쪽에 바늘을 삽입합니다. </li><li> 코의 지역 왼쪽 부근 오른쪽 바늘을 삽입하고 오른쪽 눈 속에있는 슬릿 방향으로 머리를 밀고, 부드럽게 왼쪽 눈 위에 표피를 놓으면 왼쪽 바늘을 사용합니다. 두뇌는 종종 광학 이오 모두 상대적으로 그대로, 오른쪽에있는 슬릿에서 등장한다가 없어요 역시 여전히 붙어 때때로 색소 안구 조직. </li><li> 새로운 멸균, 35mm 페트리 접시에 멸균 생리 해부 한 방울로 바늘과 전송의 뒤쪽에 머리를 밀어. </li><li> 각각의 유전자형에 대한 10-15 두뇌를 수집합니다. </li></ol> VI. 광학 엽 (叶)의 제거 및 효소 처리 <ol><li> 각 손에 27 게이지 바늘로 1 CC의 주사기를 잡고 해부 솔루션의 드롭 중 하나를 작은 영역에있는 모든 두뇌를 수집하는 이들을 사용하여 </li><li> 문화 나머지 조직은 중앙 두뇌 지역하므로 각 두뇌의 광 엽 (叶)을 제거하는 도구를 절단과 같은 주사기를 사용합니다. </li><li> 무균 papain 솔루션 1 ML를 포함 eppendorf 튜브에 하나의 유전자형의 모든 두뇌를 전송할 5 μl에서 설정 20 μl pipetman를 사용합니다. </li><li> 뇌는 60 RPM에서 회전 세트 10~15분에 대한 papain에 incubated. </li><li> 3 분 3,000 rpm으로 원심 분리기. </li></ol> 무균 층류 후드 안쪽 - 조직이 공기에 노출되어있는 모든 단계의 나머지 단계에 대한 층류 후드에서 수행되어야합니다. VII. 세탁 <ol><li> 효소 솔루션을 제거하고 멸균 해부 솔루션으로 대체합니다. </li><li> 3 분 3,000 rpm으로 원심 분리기. </li><li> 무균 해부 솔루션으로 3 회 총 씻으십시오. </li><li> 해부 생리를 제거하고 DDM2로 바꿉니다. </li><li> 원심 분리기와 DDM2 2 회 총 씻으십시오. </li><li> 멸균 35mm 페트리 접시에 전송 두뇌와 미디어. </li></ol> VIII. 해부 현미경으로 분쇄 및 도금 <ol><li> coverslip의 중심에 DDM2 5 UL 놓습니다. </li><li> 노란 팁과 DDM2이 드롭 1 머리를 전송합니다. </li><li> 현미경, 작은 조각 (&lt;1 분 소요됩니다)에 두뇌를 주사위 27 게이지 바늘을 사용합니다. </li><li> 영상지도 아래, 큰 조각이 부드럽게 pipet의 일각에 빨려하고 중간에 다시 퇴학 수 있도록 좋은 지점으로 뽑아왔다 유리 pipet의 팁을 휴식. 우리는 100 μl 혈소판 모세관 유리 표준으로 입 흡입 튜브를 사용합니다. 참고 : 미디어의 거품을 끌지 않기 위해 조심하고 당신이 경험적으로 사용할 수있는 최고의 크기 pipet을 결정해야합니다. 좋은 사람 당신이 무슨 30초 / 두뇌의 일부 개별 세포도 일부 대형 대단히 짧은 시간과 머리를 떼어 놓다 수 있습니다. 당신은 높은 전력이 볼 때, 뉴런의 약 10~20%는 여전히 남아있는 일부 프로세스를해야하고 여전히 일부 대형 대단히 짧은 시간있을 것입니다. 당신이 너무 많이 떼어 놓다면 모든 세포가 원형되며, 그들은 살아남지 못할 것이다 너무 많은 피해를 입었됩니다. 이 단계는 연습이 필요하고 신속하게 변경할 수있는 큰 표면 대 부피 비율과 DDM2의 드롭의 osmolality과 산도가 있기 때문에 신속하게 할 수 있어야합니다. 조직 최대한 빨리 CO 2 배양기에 배치해야합니다. </li><li> &quot;유전자형, 날짜 및 시간&quot;페트리 접시의 뚜껑에 작성합니다. </li><li> 100mm 배양 접시에있는 35mm 페트리 접시를 (서부 유럽 표준시 kimwipe)를 넣어 30 분 CO 2 배양기에서 쉬라 구. </li><li> DDM2 1.5 ML로 35mm의 요리를 홍수. </li><li> 5% CO 2 배양기 (23 ° C)에서 보육의 문화를 유지. 참고 : 냉각 CO 2 배양기에 액세스할 수없는 경우, 표준 포유류의 조직 문화 배양기를 사용하고 중 23 시원한 공간과 더위에 넣어 ° C, 또는 실험실에 넣어 온도를 해제하고 사용 주변 주위 온도를 조절하기 위해 보육의 하단 트레이에 얼음. </li></ol> IX. 수유 셀 <ol><li> CNBM/B27 : 1 일 (다음날)에서 3시 1분 DDM2를 만들기 위해 CNBM/B27 0.5 ML (에어컨 미디어)를 추가합니다. </li><li> CNBM/B27 : 데이에 5 3시 1분 DDM2 미디어 1 ML를 교체하십시오. </li><li> CNBM/B27 모든 4-5일 : 3시 1분 DDM2와 함께 먹이를 세포 보관하십시오. 참고 : 문화가 아닌 시설의 연결 매체없이 재배하지만 뉴런 좀 더 깨지기 쉬운 표시 및 전기 생리학 실험에 사용하기 위해 더 어렵다 수 있습니다. </li></ol>

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	<li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , (1991).</li><li>Rohrbough, J., O'Dowd, D. K., Baines, R. A., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+bases+of+behavioral+plasticity:+Establishing+and+Modifying+synaptic+circuits+in+the+Drosophila+Genetic+System.">Cellular bases of behavioral plasticity: Establishing and Modifying synaptic circuits in the Drosophila Genetic System.</a> J. Neurobiol. 54, 254-271 (2003).</li><li>O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-gated+currents+and+firing+properties+of+embryonic+Drosophila+neurons+grown+in+a+chemically+defined+medium.">Voltage-gated currents and firing properties of embryonic Drosophila neurons grown in a chemically defined medium.</a> J. Neurobiol. 27, 113-126 (1995).</li><li>Lee, D., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+excitatory+synaptic+transmission+mediated+by+nAChR+in+cultured+Drosophila+neurons.">Fast excitatory synaptic transmission mediated by nAChR in cultured Drosophila neurons.</a> J. Neurosci. 19, 5311-5321 (1999).</li><li>Su, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+synaptic+currents+in+Drosophila+mushroom+body+Kenyon+cells+are+mediated+by+alpha-bungarotoxin-sensitive+nAChRs+and+picrotoxin-sensitive+GABA+receptors.">Fast synaptic currents in Drosophila mushroom body Kenyon cells are mediated by alpha-bungarotoxin-sensitive nAChRs and picrotoxin-sensitive GABA receptors.</a> J. Neurosci. 23, 9246-9253 (2003).</li><li>Jiang, S. A., Campusano, J. M., Su, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+mushroom+body+Kenyon+cells+generate+spontaneous+calcium+transients+mediated+by+PLTX-sensitive+calcium+channels.">Drosophila mushroom body Kenyon cells generate spontaneous calcium transients mediated by PLTX-sensitive calcium channels.</a> J. Neurophysiol. 94, 491-500 (2005).</li><li>Campusano, J. M., Su, H., Jiang, S. A., Sicaeros, B., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nAChR-mediated+calcium+responses+and+plasticity+in+Drosophila+Kenyon+cells.">nAChR-mediated calcium responses and plasticity in Drosophila Kenyon cells.</a> Develop Neurobiol. 67, 1520-1532 (2007).</li><li>Oh, H. -. W., Campusano, J. M., Hilgenberg, L. G. W., Sun, X., Smith, M. A., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+chemical+synapses+and+gap+junctions+between+Drosophila+brain+neurons+in+culture.">Ultrastructural analysis of chemical synapses and gap junctions between Drosophila brain neurons in culture.</a> Develop Neurobiol. , (2007).</li></ol>

그들이 neurites을 확장하고 기능적인 시냅스 연결을 형성 어디 배아 / 출생 후의 설치류 동물의 두뇌에서 수확 뉴런은 기본 세포 배양 재배하실 수 있습니다. 이러한 문화의 준비를위한 방법은 잘 구축하고 있으며 설치류의 연결을 문화의 연구 버렸네 형성과 기능의 조절 (뱅커와 고슬린, 1991)에 관련된 유전자와 환경 요인을 식별하는 중요한 역할을했습니다. 인류의 다양한 곤충 뉴런도 문화의 성장 수 있지만, 문...

코팅 Coverslips : 60mm 배양 접시에있는 autoclaved coverslips 놔. 절약의 Pipet 5 UL 각 coverslip의 중심에 A / Laminin 믹스. 2 시간 37 C 인큐베이터에 놓습니다. autoclaved 물을 각각 100 UL과 배 coverslips 린스. 멸균 파스퇴르 pipet에 연결된 진공을 사용합니다. 헹굼 마지막 동안, 집게로 coverslip을 선택하고 양면을 건조. 35mm 페트리 접시에 coverslip을 전송합니다. 한달에 최대 상온에서 보관하십시오.

primary neuronal cultures from the brains of late stage drosophila pupae

이 비디오에서는, 우리는 늦은 무대 Drosophila pupae의 두뇌에서 기본의 연결을 문화의 준비를 보여줍니다. 절차는 번데기 형성 후 70-78 시간에 동물의 두뇌의 제거와 함께 시작됩니다. 고립된 두뇌는 혈청이없는 성장 매체에 여러 세척 다음 papain의 짧은 부화 후 표시됩니다. coverslip에 대한 미디어의 5 UL 드롭의 각 두뇌의 기계적 분리의 과정을 그림입니다. 포스트 mitotic의 뉴런의 axons과 dendrites이 분리되는 동안 소마 근처에서 털을 깎으하지만 뉴런은 도금 몇 시간 이내에 프로세스를 생성하기 시작합니다. 이미지 2 일에서 살고 문화를 보여줍니다. 뉴런은 문화의 첫 번째 주 동안 프로세스를 정교로 진행합니다. 특정의 연결 인구는 같은 GFP 또는 RFP 같은 형광 마커의 조직 구체적인 표현을 드라이브에 GAL4 라인을 사용하는 문화에서 확인할 수 있습니다. 전체 셀 녹음은 교양 뉴런은 기능, 자발적으로 적극적인 cholinergic 및 GABAergic 시냅스를 형성 증명하고있다. 짧은 비디오 세그먼트는 교양 뉴런의 접시에 자발적인 칼슘 과도 및 니코틴 evoked 칼슘 반응을 모니터하기 위해 칼슘 표시기 색소로 Fura - 2를 사용하여 교양 뉴런의 칼슘 역학을 보여줍니다. 이러한 pupal 뇌 문화 유전 및 약리 도구 중앙 시냅스 형성과 기능에 영향을 내장하고 외부 요인을 식별하는 데 사용될 수있는 유용한 모델 시스템입니다.

Watch this Scientific Journal Video about Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae at JoVE.com

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

이 동영상은 늦게 무대 Drosophila pupae의 두뇌에서 기본의 연결을 문화의 준비를 보여줍니다. 라이브 문화의 전망 Fura - 2를 사용하여 칼슘 수준의 neurite의 파생물 및 이미징을 보여줍니다.

늦은 단계 Drosophila Pupae의 두뇌에서 기본의 연결을 문화

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

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Research

JoVE Journal

Biology

15.0K 조회수.  University of California, Irvine (UCI). 이 동영상은 늦게 무대 Drosophila pupae의 두뇌에서 기본의 연결을 문화의 준비를 보여줍니다. 라이브 문화의 전망 Fura - 2를 사용하여 칼슘 수준의 neurite의 파생물 및 이미징을 보여줍니다.

비디오: Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Midgastrula 스테이지 Drosophila의 태아에서 문화의 연결 준비

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

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생물학

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

성장 콘스의 고해상도 이미징을위한 Aplysia에서의 연결을 셀 문화

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

쥐 Neonates에서 기본 Dissociated Midbrain의 도파민 세포 문화

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

의 준비 Aplysia 감각 - 모터의 연결을 셀 문화

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

개발 및 성인의 준비 Drosophila 두뇌와 라이브 영상을위한 Retinae

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

설치류에서 기본 성인 Fibroblast 문화를 구축

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

에서 기본 셀 문화 Drosophila Gastrula의 태아

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

인간의 기본 섬유 아세포 배양에서 순진 성인 줄기 세포 분리

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

의 유틸리티 스테이지 별 중후반<em&gt; 초파리</em&gt; 난포 격리

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...