우리는 이미지 처리 및 분석 조언 데니스 워드 컨트리의 .. 감사합니다, 데이비드 W. Silversides 그의 실험실에서 Gata4p [5킬로바이트-GFP 마우스 라인이 생성되었습니다. 필론 실험실에서 연구를 CIHR, NSERC, FRQS 및 FRQNT에 의해 투자된다.

윤리 문 마우스를 포함하는 실험은 의학 연구에 사용 된 동물의 관리 및 조작을위한 동물 보호 지침의 캐나다 협의회 다음을 수행 하였다. 동물의 조작을 포함하는 프로토콜은 몬트리올 퀘벡 대학의 기관 윤리위원회에 의해 승인되었습니다 (위원회 (CCT) Institutionnel 드 보호 데 Animaux, 참조 번호 0512-R3-650-0513). 1. 콜라겐 젤의 제조 조직 문화 후드에서 멸균 방식으로 작업 할 수 있습니다. <ol><li> 표준 항생제 등을 포함한 5 배 DMEM (이글의 수정 필수 매체)를 준비합니다. 20 ㎖의 물에 DMEM 분말과 탄산 수소 나트륨의 0.925 g의 3.37 g을 녹인다. 0.22 μm의 필터를 통해 전달하여 소독. 무균 100X 페니실린 / 스트렙토 마이신 및 멸균 열 불 활성화 된 소 태아 혈청 25 ㎖의 2.5 ML을 추가합니다. 4 ℃에서 보관 </li><li> 얼음에서 800 μl를 혼합완전한 배 DMEM 600 ㎕의 1 N NaOH를 17 ㎕의 콜라겐 I 용액 (0.02 N 아세트산, 필터 소독에 3.77 ㎎ / ㎖). 3 ㎖의 최종 부피로 멸균 수로 희석. 믹스 관련 성장 인자를 포함합니다. 우리는 일반적으로 장내 NCC 마이그레이션을 자극하는 10 NG / ㎖에서 GDNF를 사용합니다. </li><li> 24 - 웰 플레이트의 단일 행의 각 웰 예금 480 μL. 거품을 피하십시오. 나머지 행은 세포 이동의 다른 성장 인자의 효과를 테스트하기 위해 사용될 수있다. </li><li> 콜라겐은 37 ℃에서 멸균 배양기에서 1 시간 이상을 중합하자 </li></ol> 2. 동물 (16)의 해부 <ol><li> 교배를 설정하고 다음 날 아침에 질 플러그를 확인합니다. 일 E0.5는 질 플러그가 발견되는 일 정오되는 여성을 격리하며 E12.5 12 시까 지 일을 기다려야. </li><li> 이소 플루 란으로 임신 한 쥐를 마취 및 CO 2 흡입에 의해 안락사. </li><li> 70 % 에탄올로 마우스 스프레이. 복부의 리프트친척 및 해부 가위로 복부에 구멍을 엽니 다. </li><li> 얼음처럼 차가운 PBS (인산염 완충 식염수)로 채워진 유리 페트리 접시에 자궁을 제거합니다. 각각의 배아 주입 사이트를 분리하기 위해 가로 방향으로 개별 deciduum의 부종과 자궁을 잘라. </li><li> 얼음처럼 차가운 PBS로 가득 다른 유리 페트리 접시에 따로 따로 각 주입 사이트에서 작동합니다. 해부 현미경 하에서 자궁 근육층을 제거하는 미세 집게를 사용한다. </li><li> 배아를 나타 내기 위해 내장 난황과 양막을 엽니 다. 그들이 개발 창자 고리로 연결되었다는로, 태반 / 내장 난황에 배아를 결합하는 혈관을 절단하는 경우주의하십시오. </li><li> 목에서 배아의 머리를 절단. </li><li> 그냥 어두운 붉은 색, 간 이상, 태아의 복강에 닫힌 집게를 삽입하고 복부 구멍에 횡단 구멍을 만들기 위해 (압력을 적용 중지) 자체의 집게가 열려하자. 향해 열린 집게를 아래로 당겨완전히 복부를 열 수있는 배아의 뒤쪽 끝. </li><li> 간 뒤에 결합 조직을 잡고 (콜론 항문에 부착) 내장을 파괴하지 않도록주의하고, 복부 밖으로 내장을 빼냅니다. </li><li> 배아의 나머지 부분에서 용기를 무료로 결장을 잘라. 컷 콜론의 임의의 위치에서 할 수있다. 이후의 꼬리 부분을 예약. </li><li> 맹장의 결합 조직, 내장의 다음 나머지를 애타게. 그렇게하는 동안 장내 상처하지 않도록주의하십시오. </li><li> 약간의 위험이있는 경우에는 간, 위 (소장의 주동이의 끝)뿐만 아니라 mesonephros 성기 능선을 버려야. </li><li> 소장을 분리합니다. 또, 대장을 잘라 내지 않도록주의하십시오. 지금부터, 창자 조직의 rosrrocaudal 방향은 주동이의 끝에 존재하는 날카로운 곡선을 사용하여 추적 할 수 있습니다. EM 전에 가능한 한 짧은 시간 동안 실온에서 PBS에 소장 남기기침구 (단계 3.3 참조). </li><li> 마지막으로, 정확하게 배아 발달의 단계를 결정하기 위해 각각의 배아에 대한 꼬리 somites의 수를 기록한다. </li></ol> 3. 배아 창자의 단면 <ol><li> 배아 해부 진행하기 전에 100 ㎖의 PBS (인산염 완충 식염수)에 1.5 g 아가로 오스를 녹여 50 ℃로 유지 </li><li> 부어 삽입 금형에 용융 아가로 오스 (예를 들어, 약 1 / 4 튜브 벽의 절제하는 길이 절단 된 2 ML의 microcentrifuge 관을 폐쇄). 아가로 오스는 약 42 ~ 45 ° C의 열을 식히, 그것은 터치에 약간 따뜻해야한다. </li><li> 다만 아가로 오스 응고하기 전에 배아 소장을 포함 (이것은 집게 팁으로 평가 될 수 있으며, 주변에 36 ~ 38 ° C 발생)을. 접힌 주동이의 말에 집게로 장을 들고, 금형의 길이를 따라 아가로 오스를 통해 매우 천천히 잡아 당깁니다. 이것은 바로 아가 세트 동안 창자를 유지하는 데 도움이됩니다. T 릴리스즉시 이동하고 저항하기 시작 실행합니다. 창자의 주동이의 - 꼬리 방향을 확인합니다. </li><li> 아가로 오스가 완전히 설정 한 보장하기 위해, 냉장고 2-3분에 금형을 넣어. </li><li> (열린 에펜 도르프 튜브 밖으로 밀어) 금형에서 아가로 오스를 꺼내. 블레이드, 소장에 수직 커트를하고, 양쪽 끝에서 초과 아가로 오스를 꺼내. </li><li> 진동 블레이드와 마이크로톰의 금속 무대에서 아래로 차단 아가 소장 /의 주동이의 끝을 붙입니다. 소장 / 아가로 오스 블록의 측면에 여분의 아가로 오스를 낸다. </li><li> 진동 마이크로톰 챔버의 금속 단계를 탑재합니다. 필요에 따라서는 소장 (따라서 블레이드에 수직) 가능한 한 수직 스테이지의 각도를 조정한다. 얼음처럼 차가운 PBS로 표본을 커버. 버퍼 표면에서 몇 mm까지 마이크로톰 블레이드를 가져옵니다. </li><li> 해당 전자를 보장 꼬리 가장 소장의 200 ㎛ vibratome 가로 컷을 만드는ACH 아가로 오스 조각은 전체 장 섹션이 포함되어 있습니다. </li></ol> 4. 장내 외​​식 문화 <ol><li> 부드럽게 잘 각각의 중앙을 향해 한 조각을 배치, 집게로 콜라겐 젤에 갓 잘라 소장 / 아가로 오스 조각 평면을 예금. </li><li> 이식편 중 NCC의 마이그레이션을 할 수 있도록, 습도 5 % CO 2 분위기에서, 37 ° C 3 일간 배양한다. </li><li> 집게 매우 부드럽게 콜라겐 겔 떨어져 소장 / 아가로 오스의 조각을 가져 가라. 아래의 젤이 손상되지 않도록주의하십시오. </li><li> 세포의 이동 패턴이 교란되지 않도록하기 위해 후속 배양 및 세척 단계에서 직접 콜라겐 겔을 만지지 마십시오. 실온에서 잘 1 시간 당 (PBS에서) 4 % PFA (파라 포름 알데히드)의 500 μL로 수정합니다. </li><li> 물론 당 DAPI 500 μL (4 &#39;, 6 - diamidino -2 - 페닐 인돌) 솔루션 (PBS에서 5 ㎍ / ml)로 정착을 교체하고 실온에서 10 분 알을 품다. </li><li> 잘 배 각각의 세척5 분 동안 PBS 500 μL와. </li><li> 각 웰 내의 콜라겐 겔에 포함 된 형광 세포 (GFP 및 DAPI 채널)의 사진. </li></ol> 5. 이미지 분석 우리는 이식편 배양 한 후 생성 된 이미지를 처리하고 정량화 ImageJ에 17를 광범위하게 사용했다. <ol><li> 형광 세포 사진과 같은 배율에서 마이크로 미터 슬라이드 이미징에 의해 시작합니다. (직선 도구를 사용하여) 픽셀 1 미크론의 길이를 측정한다. </li><li> (/ 설정 규모를 분석, 픽셀 / 마이크론의 입력 번호) 규모를 설정합니다. </li><li> 각 GFP의 형광 사진을 위해, 8 비트 그레이 스케일 (Image/Type/8-bit)로 이미지 형식을 변경합니다. </li><li> 신호 (이미지 / 조정 / 밝기 / 명암 ​​대비)의 강도를 조정합니다. </li><li> 필요 (프로세스 / 배경 빼기, 롤링 볼 반경을 조정)하면 배경 잡음을 뺍니다. </li><li> 세포와 세포 덩어리 (이미지 / / 임계 값을 조정), 강조하기 위해 임계 값을 설정합니다N 덩어리 (공정 / 진 / 유역을) 분할하는 분수령을 적용합니다. </li><li> 관심 영역 (ROI)을 지정하고 세포 수의 통계를 생성하는 입자를 분석 (입자 분석 / 분석, 설정 크기 최소 남아있는 픽셀을 제외). </li><li> 의 Feret의 직경 (설정 / 측정 분석)를 포함하는 측정 옵션을 설정합니다. </li><li> 그룹의 ROI와의 Feret의 직경을 결정하기 위해 전체로 측정, 세포의 확산의 표시 (/ 도구 / ROI 관리자 / 더 / 또는 다음 ROI 관리자 추가 / ROI 관리자 / 측정 분석). </li></ol>

<ol>
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저자가 공개하는 게 없다.

우리는 우리의 생체 이식편 배양 기술은 정확하게 GDNF의 존재에 장내 NCC 이주 가능성을 정량화하는 데 사용할 수있는 방법을 보여줍니다. 이러한 정확한 정량이 크게 11,12,15 이전에 설명 된대로, 200 ㎛ 두께의 vibratome 창자 부분을 사용하는 대신 대략적인 크기의 큰 조각에 의해 촉진된다. 사실, 이것은 우리가 재현성이 높은 환경에서 세포의 적절한 수와 함께 작업 할 수 있?...

신경 능선 세포 (NCC)의 배아를 개발하는 동안 많은 유도체를 형성하는 척추 동물에 고유 한 과도 세포 유형입니다. 이 세포 집단이 아닌 신경 외배엽 1에 인접한 신경 판의 경계에서 발생한다. neurulation 동안, 성형 신경관의 등쪽 가장자리를 따라 신경 판의 장소 NCC의 굽힘. NCC는 분리하는 멀리 신경 튜브에서 마이그레이션, 상피 - 간엽 전환을 받고있다. NCC들은 전체 장용 신경계 (ENS)를 형성 소화관 장벽에 내장 신경 신경절의 상호 연결된 네트워크를 포함하는 다양한 배아 구조를 정착. 최근 2,3 리뷰, 많은 유전자는이 복잡한 구조의 개발에 참여하고있다. ENS의 대부분은 (즉, 미래의 후뇌 / 척수 경계 정도) 미주 신경 튜브에서 NCC의 원래의 작은 수영장에서 파생 4.이 신경 전구 세포는 쥐의 배아 일 (E) 9.0 주위에 foregut에 도달 약 e15.0 전체 배아 창자를 식민지 때까지 용기의 중간 엽에서 꼬리 쪽 마이그레이션 할 수 있습니다. 대장의 신경 전구 세포의 작은 부분 집합은 최대 맹장 4 반대 방향으로 후방 창자 침입 성례 NCC에 의해 제공됩니다. 미주와 성례의 두 NCC가 필요 여러 마이그레이션, 핵 확산, ENS의 완전한 형성을 보장하기 위해 생존과 분화 촉진 단서.을 이와 관련, 동물 모델 - 특히 유전자 변형 생쥐는 - 여러 가지 중요한 세포 외 리간드의 식별 수단이되었습니다 GDNF (아교 세포 유래 신경 영양 인자), 엔도 텔린-3, 뉴로-3, BMP에 (뼈 형태 형성 단백질), 네트 린에게 뿐만 아니라, 음과 인도의 고슴도치 (쉬와 IHH) ~ 10. 이들 중, (형질 전환시 재 배열) 티로신 키나제 횡단 수용체 RET을 통해 신호 GDNF는 번째로 인식되고배아 창자 및 내 매력과 NCC의 지침에 대한 전자의 가장 중요한 통로. GDNF는 창자의 중간 엽에서 분비 및 RET (11, 12)을 표현 장내 NCC, 직접 chemoattractive 인 시공간으로 제어 rosrrocaudal 구배를 형성한다. 다른 기능 사이에, ENS는 장 벽에 부드러운 근육과의 상호 작용을 통해 소화 기관 내에서의 움직임을 조절한다. Hirschsprung의 질병에있는 장 결과의 말단 부위에있는 신경 중추의 부재 : 영향을받는 부분의 토닉 수축 막힘, 소화 물질의 상류 축적과 창자와 복부의 거대한 팽창에 이르게한다. Hirschsprung 병은 약 5,000 정상 출산을 발생합니다. 장내 NCC의 rostro - 꼬리 이주 패턴은 Hirschsprung의 질병의 원인에 대한 주요 기여 요인이 될 것으로 생각됩니다. NCC와 B의 마지막 부분을 마이그레이션하는 소스의 결장, 먼정착 할 owel는 ENS 형성의 결함에 가장 민감하다. 장내 NCC 마이그레이션에서의 중요한 역할에 따라, GDNF / RET 신호의 중단 Hirschsprung의 질병 (13)의 주요 알려진 유전 적 원인이다. [5킬로바이트-GFP 14 Gata4p 이름 - - 철새 NCC는 녹색 형광 단백질 (GFP)으로 표시되는 더 나은 NCC와 ENS 개발을 연구하기 위해, 우리는 유전자 변형 마우스 라인을 생성합니다. 우리는 다음에 지금과 같은 GDNF 등의 다양한 성장 인자의 존재에 장내 NCC 마이그레이션 가능성의 정확한 정량화 할 수 있습니다 다른 그룹 11,12,15에서 출판 작업에서 적응 생체 세포 이동 분석을, 완성.

<table border="1">
	<tbody>
		<tr>
			<td>DMEM powder</td>
			<td>Wisent</td>
			<td>219-010-XK</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Bioshop</td>
			<td>SOB999</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr>
			<td>Steriflip vacuum filtration system (0.22 micron)</td>
			<td>EMD Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin solution, 100x</td>
			<td>Wisent</td>
			<td>450-201-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Wisent</td>
			<td>095-150</td>
			<td>High quality grade</td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>BD biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Bioshop</td>
			<td>SHY700</td>
			<td>Diluted from 10 N stock then sterile-filtered</td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Cedarlane</td>
			<td>CLCYT305</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 24-well Plate</td>
			<td>BD biosciences</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>Fisher Scientific</td>
			<td>089515</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri dish</td>
			<td>VWR</td>
			<td>89000-306</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td>Cell culture grade</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica</td>
			<td>M125</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioshop</td>
			<td>AGA001</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Feather</td>
			<td>21</td>
			<td></td>
		</tr>
		<tr>
			<td>All Purpose Instant Krazy Glue Pen</td>
			<td>Krazy Glue</td>
			<td>KG824</td>
			<td></td>
		</tr>
		<tr>
			<td>HM 650V Vibrating-Blade Microtome</td>
			<td>Thermo Scientific</td>
			<td>920110</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9564</td>
			<td></td>
		</tr>
	</tbody>
</table>

a quantitative cell migration assay for murine enteric neural progenitors

신경 능선 세포 (NCC)는 등의 신경관에서 유래 및 개발 척추 동물의 배아에 걸쳐 광범위하게 마이그레이션 과도 다 능성 세포 집단이다. 말초 뉴런 및 아교를 제공 할뿐만 아니라, NCC는 멜라닌 세포뿐만 아니라 부교감 얼굴 골격의 대부분을 생성한다. NCC 마이그레이션 및 차별화 신경 튜브 및 지역에 별개의 세포 외 신호에 대한 노출을 따라 자신의 축 원점의 조합에 의해 제어된다. 세포 외 리간드의 이러한 기여는 장용 신경계 (ENS), 복잡한 상호 연결 (다른 것들 사이에) 로컬로 제어하는​​ 신경 중추의 네트워크 창자 근육 운동과 장 운동의 형성시에 특히 분명하다. 꼬리 패션에 주동이의 - - 미래의 창자의 전체 길이를 ENS의 대부분은 정착하기 위해 긴 여행을 수행 NCC의 작은 초기 수영장에서 파생됩니다. 로 알려진 몇 가지 신호 전달 경로 중장내 NCC의 식민지에 영향을 미치는, GDNF / RET 신호가 가장 중요한 것으로 인식되고 있습니다. 사실, 창자의 중간 엽으로 RET 리간드 GDNF의 시공간으로 제어 분비 배아 창자 및 내 매력과 RET - 표현 장내 NCC의 안내를 주로 담당하고 있습니다. 여기, 우리는 GDNF 등 다양한 성장 인자의 존재에 장내 NCC 마이그레이션 가능성 정확한 정량화 할 수 찬란 NCC를 가진 유전자 변형 마우스 라인을 사용하고, 생체 세포 이동 분석에 대해 설명합니다.

다음과 같은 결과 (그림 1) 여기에 설명 된 기술로 얻을 수있는 대표입니다. 성장 인자 (즉, GDNF)의 사용은 GFP를 발현하는 장 이식편에서 그리고 콜라겐 겔에 장내 NCC (그림 2)의 이동을 자극한다. 일부 세포가 성장 인자의 부재하에 이식편 밖으로 나와 있지만, 이들은 주로 GFP-표지 아니며 패시브 엔트리를 나타낸다. 그것은이 조직이 여전히 크게 형광 세포에 의해 채...

Watch this Scientific Journal Video about A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors at JoVE.com

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

우리는 다양한 성장 인자의 존재 하에서 장용성 신경 능선 세포 이동 전위의 정확한 정량을 허용 생체 세포 이동 분석을 제시한다.

쥐과 장내 신경 전구 세포에 대한 양이 세포 이동의 분석

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

a-quantitative-cell-migration-assay-for-murine-enteric-neural

Research

JoVE Journal

Neuroscience

9.2K 조회수.  UQAM.  우리는 다양한 성장 인자의 존재 하에서 장용성 신경 능선 세포 이동 전위의 정확한 정량을 허용 생체 세포 이동 분석을 제시한다. 

비디오: A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

만성 두 광자를위한 박막 해골 창 기법 생체내에서</emNeuroinflammation의 모델 Murine Microglia의> 이미징

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

연구

신경과학

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Neurosphere 분석을 사용하여 마우스 배아 신경 줄기 세포 문화 구축

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

세포 어세를 형성 신경 콜로니 : 신경 전구 세포로부터 타고난 신경 줄기 세포를 차별 어세이

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

수정 Zigmond 회의소 분석을 사용하여 트렁크 신경 크레스트 세포 마이그레이션 분석

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

크로스 종 이식에 의한 신경 크레스트 마이 그 레이션과 차별화 분석

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neuroblast 분석 : 유동세포계측법을 사용하여 차별화 신경 줄기 세포에서 자손의 연결 전구 세포의 생성과 강화에 대한 분석

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

배아 Murine 신경 튜브에서 신경 크레스트 세포의 분리 및 문화

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

외부 응용 직류 전기 분야에 신경 전구체 세포 마이그레이션 속도론 분석을위한 Galvanotaxis 분석

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

쥐의 눈으로 인간 망막 안료 상피 세포의 트랜스 scleral Subretinal 이식에 대 한 세련 된 프로토콜의 개발

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

절연 및 신경 창시자 Chromatin-Immunoprecipitation 히스톤 3 리 79 Dimethylation 마크의 뒤의 재배

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...