이 연구는 텍사스 A &amp; M 대학에서 시작 펀드로 일부 자금되었습니다

절개 전에 준비 수술 도구 : 압력솥의 모든 해부 가위와 집게를 소독 Coverslip 청소 : <ol><li> 최소 24 H에 대한 33% HCL에 1L 잔디 비커의 모든 coverslips을 (24 잘 접시 직경 15mm) 소크 </li><li> 모든 잔류 HCL을 제거 가끔 흔들어 10 분 물을 실행하는 와시 </li><li> Millipore 물로 헹굼 </li><li> 물을 배수하다 </li><li> 95~98% 에탄올에 coverslips 소크 </li><li> 플랫 플레이트와 공기의 깨끗한 조직 전송 coverslips은 coverslips을 건조 </li><li> 알루미늄 호일과 압력솥와 커버, 유리 비커에 coverslips을 전송 참고 : Coverslips이 단계에서 저장할 수 있습니다 </li><li> 문화 접시에 개인 coverslip를 놓습니다. 코팅 준비 </li></ol> Coverslip 코팅 : <ol><li> PBS 및 필터 - 소독 (0.22 μm의)와 폴리 - D - 라이신 100 X 주식 (PDL, -20 ° C에서 aliquots로 저장 PBS에서 알부민 0.5 % 소 혈청에서 10 MG / ML을) 희석 </li><li> 37 2 H 1 X PDL 솔루션 (~ 0.15 ML / cm 2) · 인큐베이터에서 C와 외투 coverslips </li><li> PDL 솔루션을 제거하고 완전히 멸균 ddH 2 O 건조 coverslips 3 번 씻어 </li></ol> 참고 : coverslips / 각 해부에 대한 번호판 충분한 문장. PDL - 코팅 coverslips 4에서 몇 주 동안 저장할 수 있습니다 ° C. 미디어 솔루션 해부 매체 (DM)는 10 MM의 HEPES (Invitrogen 15,630)과 보충 (HBSS, Invitrogen 14175) 무균 얼음 차가운 행크의 밸런스드 소금 솔루션 (W ​​/ O 칼슘 2 +, MG 2 +)를 사용합니다. 누계 문화 미디어 : 2% B27, 1 X 사토, 0.5 MM 나트륨 pyruvate, 0.75mM GlutaMAX, 60μg/ml N - acetylcysteine, 5 μg / ML : DMEM/NBB27 매체 DMEM / Neurobasal를 (권 1시 1분 권)를 포함 인슐린, 10nM D - 비오틴, 1 % 페니실린 / 스트렙토 마이신. 집계 문화 매체의 100 ML하려면 50 ML DMEM (W / O pyruvate / 글루타민, Invitrogen 11,960) 50 ML Neurobasal 매체 (Invitrogen 211034), 375 μl GlutaMax (100 X, Invitrogen 35,050), 5.5 MG 나트륨 pyruvate를 섞어 (시그마 P2256), 2 ML B27 (Invitrogen 17,504)는, 6.3 MG N - acetylcysteine​​ (시그마 A8199), 사토 주식 1 ML, D - 비오틴 주식 (4000 X 주식 40 25 μl를 (100 X 주식은 아래 참조) PBS에서 μm의는 -20에서 aliquots ° C. 시그마 B4639), 인슐린 100 μl (1000 X 주식, -20에서 aliquots로 저장 0.01N HCL 5 MG / ML ° C, 시그마 16,634), 1 ML 페니실린로 저장 / 스트렙토 마이신 (100 X 주식, Invitrogen 15140)은, 필터 소독 및 저장 4 ° C. 사토 100 X 주식 솔루션 : 주식의 40 ML을하려면 다음과 같이 혼합 400가, 400 밀리그램의 BSA (시그마 A9647), progesterone 10 μl (25 MG / ML 에탄올은 같은 MG APO - 트랜스페린 (시그마 T2252) 저장된 40 ML의 Neurobasal -20에서 aliquots ° C. 시그마 P8783), 64 MG putrescine (시그마 P7505), 그리고 나트륨 selenite (-20에서 aliquots로 저장 PBS에서 30 μm의, ° C, 시그마 S5​​261) 40 μl. -20 ° C.에 aliquots로 사토 재고 솔루션, 그리고 상점을 소독 필터 신경 도금 매체 (PM) : Neurobasal 매체, 2퍼센트 B27, 2mM 글루타민 (100x 주식, -20 ° C, Invitrogen 25,030에 저장된 aliquots), 25μM 글루탐산 (100 주식, -20 ° C에 저장된 aliquots), 1 % 페니실린은 / 스트렙토 마이신. 신경 세포의 문화 매체 (CM) : Neurobasal 매체, 2퍼센트 B27, 2mM 글루타민 (100x 주식, -20 ° C에 저장된 aliquots), 1 % 페니실린 / 스트렙토 마이신. 5 FdU 주식 : Neurobasal 매체 100x 재고, 1mM 5 - 플루오로 - 2&#39; - deoxyuridine (시그마 F0503) and1mM 유리딘 (시그마 U3003). 필터 -20 ° C.에 살균 및 aliquots로 저장 Papain의 소화 솔루션 (이전 해부 신선한 확인) : <ol><li> 4ml DM의 3.2 MG L - 시스테인 (시그마 C - 7352)를 디졸브 </li><li> 37 1N NaOH (산도 검사 스트립에서 테스트)와 물을 욕조에 장소 주변 7.4으로 산도를 조절 ° C 참고 : 오른쪽 조직 소화하기 전에 다음 단계를 (아래 참조) 수행합니다. </li><li> 20 단위 / ML의 최종 농도 papain 추가 </li><li> (0.22mm) 소독 필터, 그리고 37 물 목욕에서 개최 ° C </li></ol> 트립신 억제제 솔루션 (이전 해부 신선한 확인) : <ol><li> 20ml DM 0.2 g 트립신 억제제 (시그마 T7295)를 디졸브 </li><li> 산도를 확인하고 1 N NaOH로 ~ 7.4으로 산도를 조절 </li><li> 소독 필터, 그리고 37 물 목욕에서 개최 ° C </li></ol> 뇌 해부 설정 : <ul><li> 소독 집게와 가위 </li><li> 70 % 에탄올 </li><li> 클린 조직 종이와 diap벤치에 어 패드 </li><li> 벤치에서 자궁과 태아를위한 얼음처럼 차가운 DM과​​ 10cm의 페트리 요리 </li><li> 해부 현미경에서 얼음 플랫폼 </li><li> 얼음 DM과 2-3 6cm 요리 </li><li> 37 ° C의 물을 욕조에 따뜻한 PM </li></ul><ol><li> 임신 스프 라그 - 돌리 귀하의 기관 애니멀 케어의 승인 절차에 따라 쥐 (E16) 우리위원회 (IACUC)를 안락사 </li><li> 복부에 기저귀 패드와 ...의 난소를 제거하다 알코올에 쥐하다 </li><li> V 모양의 절개는 피부를 절단하기 위해 복부 피부와 가위를 잡아 핀셋을 사용하여 </li><li> 피부를 따로 확산과 근육 계층을 통해자를 다른 소독 핀셋과 가위를 사용 </li><li> 배아 주머 니나의 사슬을 추출하고 얼음처럼 차가운 DM을 포함 10cm 접시에 그들을 전송 </li><li> microdissection 가위를 사용하면, 신중하게 각각의 배아에서 색 및 뇌 각 배아를 제거하고, 장소는 얼음 플랫폼에 DM과 함께 깨끗한 10cm 접시에 두뇌를 해방 </li><li> 판상 후드와 해부 현미경 아래 midbrain / hindbrain 섹션을 제거하고 meninges를 제​​거하는 미세 집게를 사용하여 얼음 팩 DM으로 새 6cm 접시에 청소 cortices / 해마를 전송 참고 :이 단계에서, 조직은 효소 분해를위한 준비가되었습니다. </li><li> papain과 트립신 억제제 솔루션과 필터 소독 만들기 </li><li> cortices에서 DM 초과 / hippocampi을 제거 </li><li> 준비 papain의 소화 솔루션의 4ml 추가 </li><li> 50ml 튜브에 모든 내용을 전송 37에서 물을 욕조에 팔콘 튜브를 삽입 ° C 정확히 5 분 </li><li> 피펫과 papain 솔루션을 제거 </li><li> 2-3 분 5ml 트립신 억제제 솔루션, 소용돌이 관, 37에서 물이 욕조에서 개최 ° C 추가 </li><li> 트립신 억제제 솔루션을 제거 </li><li> 단계 13 14 세 번 반복 </li><li> 20ml 따뜻한 PM 추가 </li><li> 모든 대단히 짧은 시간이 (~ 20-30 회)까지 사라졌습니다 씹다 </li><li> 7min에 대한 100xg에서 원심 분리기 </li><li> 10ml PM의 펠렛을 Resuspend 및 원심 분리에 의해 두 번 씻어 </li><li> 일반적인 신경 세포 배양 또는 총체적 문화 NBB27/DMEM에 10ml PM에 펠릿을 Resuspend </li><li> 70μm 셀 sieves 통해 세포 패스 </li><li> 라이브 세포를 카운트 </li><li> 일반적인 신경 세포 배양을위한 세포를 아래에 설명된이나 접시 등 총체적 문화로 이동 참고 :이 단계에서 매우 풍부한 신경 세포 배양은 실험 목적에 따라 2 200-640 셀 / mm의 밀도에서 PDL - 코팅 접시에 dissociated 세포를 도금하여 얻을 수 있습니다. 세포 (~ 2 4h) 첨부 후, 따뜻한 PM과 매체를 바꿉니다. 일 4 시간 연장 기간에 대해 culturing 경우, 따뜻한 CM과 함께 매체의 절반을 교체하십시오. 고도의 연결을 문화, mitotic 억제제를 정화를위한 5 FdU (최종 농도 10 μm의)이 (DIV2 - 3 동안 즉,) 비의 연결을 세포 증식을 억제하기 위해 DIV2에 추가할 수 있습니다. 2 일 CM에서 복구 후에, 세포는 맥박이 완전한 매체 변경 다음 다른 이일 (DIV 6-7) 5 - FdU로 다시 처리됩니다. 그 이후, 신선한, 따뜻한 CM 모든 3~4일과 매체의 절반을 교체하십시오. 최대 4 주 동안 뉴런이 가능한 있습니다. </li></ol> 합산 준비와 도금 <ol><li> 2x10 6 셀 / ML의 밀도에 1X 사토 10ng/ml CNTF 및 10μM forskolin을 포함 NBB27/DMEM 매체에 dissociated 세포를 일시 중지 </li><li> uncoated 6 잘 플레이트의 각 우물에 세포 현탁액의 2ml을 전송 </li><li> 3 박에 대한 문화. 매일, 부드럽게 한번 P1000 Pipetman를 사용하여 셀을 resuspend 참고 : 세포 집합체 (그림 1)을 형성 시작합니다. </li><li> 집계 도금 전날에 코팅 플레이트 / Matrigel과 coverslips : <ul><li> 주식 Matrigel (성장 인자는 감소, BD Biosciences 354230) 얼음 추위 DMEM과 1시 20분을 희석 참고 : Matrigel의 aliquots가 제조 업체 절차에 따라 작성되어야합니다. 모든 팁, Eppendorf 튜브 및 재고 aliquots을 만들기위한 솔루션 Matrigel 중합을 피하기 위해 추운해야합니다. Matrigel의 Aliquots는 -80 ° C에서 보관 및 4 ° C.에 해동 아르 </li><li> 37 ° C 배양기에서 하룻밤 희석 Matrigel 솔루션의 300μl/well와 코트는 이전 PDL - 코팅 coverslips </li><li> 따뜻한 PBS로 세척 후 살균 따뜻한 ddH 2 O와 접시를 세척하고 인큐베이터에있는 접시를 두십시오. </li></ul></li><li> 골재 형성 후 3 일, 부드럽게 50ml 팔콘 튜브에 200μm 메쉬를 통해 중지를 다시 세포를 resuspend 및 체. 집계 중력 (~ 3 - 5 분​​)에 의해 튜브의 바닥에 정착하도록 허용 </li><li> 조심스럽게 뜨는을 제거 </li><li> 미디어를 추가 부드럽게 집계를 resuspend하고 다시 쉬라 구. 죽은 세포, 개별 세포와 찌꺼기를 제거하려면이 절차를 여러 번 반복합니다. 뜨는 아직 집계되지 않은 세포와 debri를 포함 여부를 확인하도록 현미경을 사용하여에스 </li><li> 부드럽게 같은 NBB27/DMEM 매체에서 집계 (그림 1) resuspend 및 집계 계산 </li><li> 25-30 주위 aggreggats/50ul로 집계의 밀도를 조절 </li><li> 도금에 대한 2ml Eppendorf 튜브에 집계 정지 전송 aliquots (500 1000μl) 참고 : 집합체가 튜브의 바닥에 정착하는 경향이 있기 때문에 그것이 도금에 대한 총체적 정학의 여러 튜브를 만들기 위해 매우 중요합니다. 부드럽게 자주 각 coverslip에 도금 집계 비슷한 번호를 보장하기 위해 코팅 coverslips 또는 문화 접시에 그들을 심는 전에 집계를 resuspend. </li><li> 인큐베이터에서 Matrigel - 코팅 coverslips을 포함하고있는 문화 판을 가져가라. 각 우물에서 솔루션을 제거, 따뜻한 PBS 및 ddH 2 O와 coverslips을 씻어 후 건조 짧게. 그들은 골재 시딩을 위해 지금 준비가되어 </li><li> 그들이 시딩 전에 균일하게 배포할 수 있도록 집계 중지를 포함한 튜브를 반전. 각 coverslip의 중심에 집계 정지 50μl를로드하고, 부드럽게 집계는 coverslip에 균일하게 연결할 수 있도록 더 이상 방해하지 않고 인큐베이터에 다시 접시를 넣어 참고 : 집합체의 밀도는 매우 중요합니다. 집합체가 씨앗을 품고있다면 너무 서로 가까이에 또는 시딩 밀도가 높은 경우, 그들의 성장으로 함께 병합하는 경향이있다. </li><li> 나중에 또는 이른 다음날 아침마다 잘 4-6 H에 추가 DMEM/NBB27medium (500 μl)를 추가 </li><li> 집계 문화를 유지하려면, 반은 모든 3~4일 매체를 변경합니다. 매체를 변경하면, 집계를 방해하지 않도록주의하고 이것은 부드럽게 잘 측면 벽을 따라 미디어를 추가하여 이루어집니다. 참고 : 몇 시간 판에 부착된 집계 후 집계에서 neurite 가지를 볼 수 있습니다. Axons 처음 2 주 동안 빠르게 성장하고 집계 (그림 2) 사이의 풍부한 연결을 형성합니다. Glial progenitors 밖으로 집계의 반경 마이 그 레이션 및 astrocytes과 성숙 oligodendrocytes로 시간이 지남에 따라 구별. </li></ol><img src="/files/ftp_upload/1304/1304fig1.jpg" alt="그림 1" /> 그림 1. 세정 후 다른 일 집계에 합산을 형성 세포의 위상 콘트라스트 이미지를 표시합니다. <img src="/files/ftp_upload/1304/1304fig2.jpg" alt="그림 2" /> 2 12일 Matrigel - 코팅 coverslips에 씨뿌린 후 집계 문화 그림 2. 위상 콘트라스트 이미지를 표시합니다.

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초기 연구는 시냅스 및 회전 - 중재 부유 집계 문화 1 성숙 myelin 형성을보고했다. 여기에 설명된 CNS의 집계 문화 시스템은 세포 개발, 마이 그 레이션 및 체외에서 분화의 분석을 촉진하기 위하여 전통적인 2D 문화의 편의와 입체 집계에 multipotent 전구 세포의 혈청이없는 성장을 결합한 제품입니다. 체제가 신경 전구체에 대한 수정하여 사용할 수 있습니다 세포 연구 및 신경 ?...

preparation of rat brain aggregate cultures for neuron and glia development studies

중추 신경계의 성숙 뉴런과 glia (CNS)에 progenitors의 recapitulates 개발과 차별화는 axo - glial 상호 작용, 속성과 multipotent progenitors의 분화, 그리고 oligodendroglial의 진행을 조사 neuroscientists위한 강력한 플랫폼을 제공하는 것이 체외 시스템 세포 및 분자 수준에서 세포 혈통. 우리는 여기 3~4주에 혈청이없는 배지에서 유지 수 있으며, 신경 세포 - glia 상호 작용과 CNS의 myelination을 연구하는 모델로 저희 연구실에서 사용되는 배아의 쥐 forebrains에서 CNS의 집계 문화 시스템을 설명합니다. 이 비디오 클립은 E16 쥐의 두뇌에서 이러한 CNS의 집계 문화를 분리하고 성장하는 방법을 보여줍니다. 또한, 동일한 뇌 해부에서 매우 풍부한 정기 dissociated의 연결을 문화가 쉽게 얻을 수 있으며, 다양한 연구에 사용 CNS의 뉴런 또는 다른 세포 공동 배양에 사용.

Watch this Scientific Journal Video about Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies at JoVE.com

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

배아 쥐의 두뇌 집합의 문화 시스템을위한 프로토콜이 설명되어 있습니다. 집계에 Multipotent progenitors 개발하고 뉴런, astrocytes 및 oligodendrocytes로 구별하실 수 있습니다.

신경 세포와 Glia 개발 연구 랫 두뇌 집계 문화의 준비

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

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JoVE Journal

Biology

12.3K 조회수.  Texas A&M University (TAMU). 배아 쥐의 두뇌 집합의 문화 시스템을위한 프로토콜이 설명되어 있습니다. 집계에 Multipotent progenitors 개발하고 뉴런, astrocytes 및 oligodendrocytes로 구별하실 수 있습니다.

비디오: Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

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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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 ...

ALS - Motor Neuron Disease: Mechanism and Development of New Therapies

Jeffrey D. Rothstein speaks about the pathology and mechanisms underlying amyotrophic lateral sclerosis or ALS, advances in ALS research, and current strategies towards the development of therapies.

ALS - 모터 신경 질환 : 새로운 치료법의 원리 및 개발

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

신경 소마와 Axons의 Compartmentalization위한 Microfluidic 장치의 제작

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

Dissociated 마우스 두피 신경 세포 배양의 준비

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

2 dGuo - 치료 Thymus 오르간 문화의 준비

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

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 ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

다중 구획 CNS 뉴런 - glia 공동 문화 Microfluidic 플랫폼

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

에서 독소 유도와 단백질 추출 Fusarium 종.</emProteomic 연구> 문화

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

Biomechanical 및 Mechanobiological 연구 랫 타일 힘줄의 준비

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...