이 작품은 방사선 종양학, 앨라배마 - 버밍햄 대학 종합 암 센터, 파이팅 어린이 암 재단과 가브리엘의 엔젤 재단 (ESY 있습니다.)의 부에서 IMPACT 상에 의해 지원되었다.

1. 세포 배양 <ol><li> 문화 neuronal 세포와이를 필요에 따라 취급합니다. </li><li> 15 ML 튜브에 수확 세포 : 기음 미디어, 인산염과 린스 (PBS, 칼슘, 마그네슘 무료) 생리 버퍼, 15 ML 튜브에 수집하고 적절한 혈청이 포함 된 미디어와 트립신을 중화, 트립신을 추가합니다. </li><li> 5 분 1,000 XG에서 봐. </li><li> 미디어를 대기음. </li><li> PBS에 세포를 Resuspend. </li><li> 5 분 1,000 XG에서 봐. </li><li> 신선한 PBS에서 미디어 및 resuspend 세포를 대기음. </li><li> hemacytometer이나 선호하는 셀 카운터를 사용하여 셀을 계산합니다. </li><li> 세포 샘플은 분석을 시작하기 바로 전에 준비해야합니다. </li><li> 모든 샘플은 자외선에서 DNA 손상을 방지하기 위해 어둡거나 노란 빛으로 처리해야합니다. </li></ol> 2. 혜성 분석 1. 슬라이드 준비 <ol><li> 1% 아가로 오스를 (1X 트리스베이스, 붕산, EDTA (에틸렌 다이아 민 테트라 초산), TB의 1g / 100 ML 녹음E) 3 분의 전자 레인지의 모든 과립가 사라 때까지. </li><li> 수영 용융 아가로 오스에 슬라이드과 측면을 닦아은 kimwipe으로 청소하십시오. 투명 필름에 공기 건조에 아가로 오스 할 수 있습니다. 이것은 저장할 수 있습니다 사전 및 슬라이드에 수행 할 수 있습니다. </li></ol> 2. 중성 혜성 분석 <ol><li> 사용하기 전에 적어도 1 시간에 ° C 4에 얼음 용해 용액 (2.5 M NaCl, 100 MM EDTA (에틸렌 다이아 민 테트라 초산) 산도 10, 10 MM 트리스베이스, 1 %의 나트륨 라 우릴의 sarcosinate, 그리고 1퍼센트 튼 X-100, 산도 10). </li><li> 2.2.2. 모든 과립이 사라 때까지 전자 레인지에 1% 낮은 녹는 점 아가로 오스는 (1X 트리스 자료의 1g / 100ml, 붕소의 산성, EDTA (에틸렌 다이아 민 테트라 초산)) 3 분 동안 녹음. 아가로 오스는 혜성 꼬리의 인공 유도를 방지하기 위해 ° C 물 목욕에서 37로 냉각해야합니다. 이상적으로, 아가로 오스는 사용하기 전에 반 시간 동안 냉각해야합니다. </li><li> 2.2.3. ML 100,000 세포가되도록 세포 현탁액을 희석. 낮은 녹는 점 아가로 오스 (37시 ° C)에있는 세포 현탁액을 결합1시 10분 (V / V), 와류 짧게의 비율과 즉시 혜성 슬라이드에 50 μl를 피펫. 샘플 영역 골고루 세포 현탁액을 확산하기 위해 피펫 팁의 측면을 사용합니다. 각 치료 그룹은 적어도 세중의에 있어야합니다. </li><li> 원은 슬라이드의 주변에 나타날 때까지 장소 30 분에 냉장고에 평면 슬라이드. </li><li> Prechill 4에 어둠 속에서 30 분이 솔루션에 용해 솔루션과 잠수함 슬라이드 ° C (또는 냉장고에). </li><li> 오프 넣어 또는 용해 솔루션을 기음과 1X 중립적 인 전기 영동 버퍼 (트리스베이스, 붕산, EDTA (에틸렌 다이아 민 테트라 초산), 1X TBE)을 추가합니다. 냉장고에 30 분 동안이 버퍼에 슬라이드를 둡니다. </li><li> 전기 챔버에서 prechilled 1X 중성 전기 영동 버퍼 (TBE), 전기 슬라이드 트레이에 장소 슬라이드를 추가 할 수 있습니다. 사람들이 전극에서 등거리되도록 슬라이드를 맞 춥니 다. </li><li> 1X 중립 전기가 슬라이드 위에 0.2 인치까지 버퍼 네요. 초과 버퍼 간 것전기와 fere. </li><li> cm 당 1 V (전극에 전극을 측정)에 전원 공급 장치의 전압을 설정하고 어둠 속에서 4 ° C (또는 차가운 방)에서 30 분에 실행합니다. </li></ol> 3. 알칼리성 혜성 분석 <ol><li> 사용하기 전에 적어도 1 시간에 ° C 4에 얼음 용해 용액 (2.5 M NaCl, 100 MM EDTA (에틸렌 다이아 민 테트라 초산) 산도 10, 10 MM 트리스베이스, 1 %의 나트륨 라 우릴의 sarcosinate, 그리고 1퍼센트 튼 X-100, 산도 10). </li><li> 모든 과립이 사라 때까지 3 분을 위해 전자 레인지에 1% 낮은 녹는 점 아가로 오스를 (1X 트리스 자료의 1g / 100 ML, 붕소의 산성, EDTA (에틸렌 다이아 민 테트라 초산)) 녹음. 그럼 적어도 30 분에 37 ° C의 물을 욕조에 좋아. </li><li> 1시 10분 (V / V), 와류 잠시의 비율로 용융 낮은 녹는 점 아가로 오스와 1 개 105 / ML (37시 ° C)에서 세포를 결합하고 즉시 혜성 슬라이드에 50 μl를 피펫. 샘플 영역 골고루 세포 현탁액을 확산하기 위해 피펫 팁의 측면을 사용합니다. 각 치료 그룹은 적어도 세중의에 있어야합니다. </li><li> 장소는 미끄러에스 평면은 30 분에 냉장고에있는 원은 슬라이드의 주변에 나타납니다 때까지. </li><li> prechilled 용해 용액과 4에 남겨 ° C를 어둠 속에서 야간에 1 시간에 젖어 슬라이드. </li><li> 용해 용액에서 슬라이드를 제거 슬라이드를 배출하고 알칼리 긴장을 풀기 단계로 사전에 잔류 세제와 소금을 제거하는 5 분을위한 차가운 중화 버퍼로 한 번 씻어. </li><li> 겔 전기 영동 챔버의 장소 슬라이드 슬라이드 위에 0.5 cm를 초과하지 prechilled 갓 만든 전기 영동 버퍼 (300 MM NaOH, 1 MM EDTA (에틸렌 다이아 민 테트라 초산),&gt; 13 PH)로 가득. 사람들이 전극에서 등거리되도록 슬라이드를 맞 춥니 다. </li><li> 슬라이드가 DNA와 알칼리 - 책임 손상의 표현의 긴장을 풀기을 할 수 있도록 어둠 속에서 30 분 동안 알칼리 버퍼에 앉아 보자. </li><li> cm 당 1 V (전극에 전극을 측정)에 전원 공급 장치의 전압을 설정하고 4시 30 분에 실행 ° C (또는 추운 방). </li></ol> 4. 고정과 역ining 셀 <ol><li> 초과 전기 버퍼를 배수 </li><li> 젖어은 RT에서 5 분을 위해 사전에 차가운 증류수에 슬라이드. </li><li> RT에서 5 분을위한 사전 차가운 70 % 에탄올에 젖어 슬라이드. </li><li> 밤새 드라이 샘플. 밝은 빛에 슬라이드를 노출시키지 마십시오. 샘플은 이전 단계에서 골을 실온에서 몇 달 동안 저장 될 수 있습니다. </li><li> 냉장고에 20 분에 Sybr 녹색 (PBS에 희석 1X)에 immersing하여 슬라이드를 청바지. </li><li> 슬라이드를 제거하고 그 어둠 속으로 완전히 건조 할 수 있습니다. 때 완전히 건조 아가로 오스는 투명이 될 것입니다. </li></ol> 4. 이미지 수집 및 분석 <ol><li> 녹색 필터 (Zeiss AxioVision)와 혜성 분석 소프트웨어 (지각 악기)를 사용하여 분석으로 설정 형광 현미경을 사용하여 이미지를 획득. </li><li> &quot;혜성의 머리&quot;(핵)를 클릭하고 소프트웨어에서 평균 꼬리 순간 DNA의 양을 포함하여 매개 변수를 계산핵. </li><li> 치료 당 최소한 200 세포를 분석합니다. </li><li> Microsoft Excel로 데이터를 내보낼 수도 있습니다. </li><li> 각 치료 그룹과 적절한 플롯 데이터 꼬리 순간을 의미 계산합니다. </li></ol> 5. 대표 결과 neuronal 세포에 혜성 분석 분석의 예는 그림 2와 그림 3에 표시됩니다. 이 경우 neuronal 세포의 방사선은 DNA 손상을 유도한다. 세포는 전기 분야의 대상이됨에 따라, DNA는 이후 혜성 분석 소프트웨어를 사용하여 분석 크기의 차이로 인해 서로 다른 속도로 마이그레이션합니다. 더 DNA 손상은 멀리 DNA는 핵에서 마이그레이션합니다. 이 이후 높은 꼬리 순간의 소프트웨어와 결과에 의해 포착 된 핵의 형광 강도를 낮 춥니 다. 표 1은 혜성 분석 분석 및 그림 4에서 대표 표를 묘사 represen를 보여줍니다tative 그래프는 혜성 분석에 의해 측정으로 방사선에 따라 neuronal 세포의 DNA 손상을 비교. <img src="/files/ftp_upload/50049/50049fig1.jpg" alt="그림 1"/> 그림 1. 혜성 분석의 흐름 차트. <a href="https://www.jove.com/files/ftp_upload/50049/50049fig1large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오</a> . <img src="/files/ftp_upload/50049/50049fig2.jpg" alt="그림 2"/> 그림 2. neuronal 세포의 대표 이미지 (A)없이와 (B) 혜성 꼬리. <img src="/files/ftp_upload/50049/50049fig3.jpg" alt="그림 3"/> 혜성 분석 소프트웨어를 사용하여 그림 3. 혜성 분석 분석. (A) 이미지의 대표 스크린 샷은 칼 Zeiss 형광 현미경을 사용하여 획득과 혜성을 사용하여 분석분석 소프트웨어. (B) 핵 또는 &quot;혜성의 머리&quot;(녹색 동그라미)을 클릭하면 형광지도와 그래프 (노란색 동그라미)와 (C) 데이터 테이블을 (빨간색 원)으로 생성합니다. <a href="https://www.jove.com/files/ftp_upload/50049/50049fig3large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오</a> . <img src="/files/ftp_upload/50049/50049fig4.jpg" alt="그림 4"/> 그림 4. 중립적 인 혜성 분석을 사용하여 방사능과 비 방사능 neuronal 세포의 DNA 손상을 분석하여 얻은 평균 꼬리 순간을 음모하여 얻은 대표 그래프. 예상대로 방사능 neuronal 세포에서 높은 평균 꼬리의 순간에 의해 묘사로 3Gy 방사선 (x-선)은 DNA 손상을 유도한다. , ** P &lt;0.01 - 평균 꼬리 순간 (표준 오류 + /)입니다 표시됩니다. <img src="/files/ftp_upload/50049/50049table1.jpg" alt="표 1"/> 표 1. </STRONG&gt; 방사능과 비 방사능 neuronal 세포에 혜성 분석 분석 얻은 대표 표. <a href="https://www.jove.com/files/ftp_upload/50049/50049table1large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오</a> .

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+sensitivity,+H2AX+phosphorylation,+and+kinetics+of+repair+of+DNA+strand+breaks+in+irradiated+cervical+cancer+cell+lines.">Radiation sensitivity, H2AX phosphorylation, and kinetics of repair of DNA strand breaks in irradiated cervical cancer cell lines.</a> Cancer Res. 64, 7144-7149 (2004).</li><li>Bonner, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[gamma]H2AX+and+cancer.">[gamma]H2AX and cancer.</a> Nat. Rev. Cancer. 8, 957-967 (2008).</li><li>Finney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+Field+Gel+Electrophoresis.">Pulsed Field Gel Electrophoresis.</a> Current protocols in molecular biology. , (2000).</li><li>Electrophoresis, F. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pulsed-field gel electrophoresis. , (2000).</li><li>Nowsheen, S., Bonner, J. A., Yang, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+poly+(ADP-Ribose)+polymerase+inhibitor+ABT-888+reduces+radiation-induced+nuclear+EGFR+and+augments+head+and+neck+tumor+response+to+radiotherapy.">The poly (ADP-Ribose) polymerase inhibitor ABT-888 reduces radiation-induced nuclear EGFR and augments head and neck tumor response to radiotherapy.</a> Radiotherapy and Oncology. , (2011).</li></ol>

관심 없음 충돌이 선언 없습니다.

혜성 분석은 개별 세포를 분석의 고유 한 능력이 있습니다. 이 세포 독성 요원에게 차등 응답을 보여 세포의 subpopulations의 식별에 유리한 것입니다. 몇 실제적인 제한이 고려되어야한다. 개별적으로 평가 될 수있는 세포의 수는 개인에 따라 다를 수 있습니다. 인구 내에서 DNA 손상에 차이가있을 경우 표본의 크기가 증가해야합니다. 괴사 또는 apoptotic 세포의 주된 존재가 에러가 추가되므로 가능한...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> 1.5 ML 튜브 </td><td> 산타 크루즈 (Santa Cruz) 생명 공학, Inc의 </td><td> SC-200271 </td><td></td></tr><tr><td> 10X TBE (트리스베이스, 붕소의 산성, EDTA (에틸렌 다이아 민 테트라 초산)) </td><td> 피셔 과학 </td><td> BP13331 </td><td></td></tr><tr><td> 15 ML 튜브 </td><td> 피셔 과학 </td><td> 0553851 </td><td></td></tr><tr><td> 아가로 오스 </td><td> 시그마 </td><td> A5093 </td><td></td></tr><tr><td> 알루미늄 호일 </td><td> 피셔 과학 </td><td> 01213101 </td><td></td></tr><tr><td> 굽​​ 달린 큰 컵 </td><td> 피셔 과학 </td><td> FB102300 </td><td></td></tr><tr><td> 원심 분리기 </td><td> 열 과학 </td><td> 75004261PR </td><td></td></tr><tr><td> 혜성 분석 소프트웨어 </td><td> 혜성 분석 IV 이미지 분석 시스템 </td><td></td><td></td></tr><tr><td> 실린더 </td><td> 피셔 과학 </td><td> 08555F </td><td></td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) </td><td> GIBCO </td><td> 15,575 </td><td></td></tr><tr><td> 전기 챔버 </td><td> 열 과학 </td><td> 09528101 </td><td></td></tr><tr><td> 에탄올 </td><td> 피셔 과학 </td><td> A407P4 </td><td></td></tr><tr><td> 형광 현미경 </td><td> Zeiss Axio 비전 </td><td></td><td> 그린 필터 가진 형광 현미경은 충분 </td></tr><tr><td> Hemacytometer </td><td> 피셔 과학 </td><td> 0267152 </td><td></td></tr><tr><td> 낮은 녹는 점 아가로 오스 </td><td> Promega </td><td> V2111 </td><td></td></tr><tr><td> 마이크로파 </td><td> 시어스 </td><td></td><td></td></tr><tr><td> 인산염은 무료 생리, 칼슘 무료, 마그네슘 버퍼 </td><td> HyClone </td><td> SH3025601 </td><td></td></tr><tr><td> 전원 공급 장치 </td><td> BioRad </td><td> 1645050 </td><td></td></tr><tr><td> 냉장고 </td><td> 시어스 </td><td></td><td></td></tr><tr><td> 지배자 </td><td> 스테이플 </td><td></td><td></td></tr><tr><td> 슬라이드 </td><td> 피셔 과학 </td><td> 12550143 </td><td></td></tr><tr><td> 나트륨 염화물 </td><td> 시그마 </td><td> S7653 </td><td></td></tr><tr><td> 수산화 나트륨 </td><td> 시그마 </td><td> S5881 </td><td></td></tr><tr><td> 나트륨 라 우릴 sarcosinate </td><td> 피셔 과학 </td><td> S529 </td><td></td></tr><tr><td> Sybr 녹색 </td><td> Invitrogen </td><td> S7585 </td><td></td></tr><tr><td> 쟁반 </td><td> FIS그녀 과학 </td><td> 15242B </td><td></td></tr><tr><td> 트리스베이스 </td><td> 시그마 </td><td> T6066 </td><td></td></tr><tr><td> 트리톤 - X 100 </td><td> 시그마 </td><td> T8787 </td><td></td></tr><tr><td> 트립신 </td><td> HyClone </td><td> SH3023601 </td><td></td></tr><tr><td> 소용돌이 </td><td> 피셔 과학 </td><td> 02216108 </td><td></td></tr><tr><td> 열탕 목욕 </td><td> 피셔 과학 </td><td> 154622Q </td><td></td></tr></tbody></table>

assaying dna damage in hippocampal neurons using the comet assay

약물의 수는 DNA 복구 경로를 타겟으로하고 세포가 DNA 손상을 만들어 죽 유도. 따라서, 직접 D​​NA 손상을 측정하기위한 프로세스를 광범위하게 평가되었습니다. 전통적인 방법은 시간이 소요, 비용, 리소스를 집중하고 세포가 복제를 필요로합니다. 반면, 혜성 분석, 단일 세포 겔 전기 영동 분석은 DNA 손상 및 수리를 빠르고 비 침습적, 저렴한, 직접 민감한 측정하기위한 한 방법입니다. DNA 손상뿐만 아니라 DNA 수리의 모든 양식이 강력한 기술을 사용하여 단일 세포 수준에서 시각화 할 수 있습니다. 혜성 분석의 기초 원리는 DNA 손상이 조직을 방해 반면에 그대로 DNA가 높은 주문이다. 손상된 DNA의 아가로 오스 매트릭스에 새어 나온 및 전기 분야의 대상이 때, 부정적인 청구 DNA가 긍정적으로 청구됩니다 음극으로 마이그레이션합니다. 큰 피해 DNA의 가닥 훨씬 핵에서 마이그레이션 할 수 없습니다. DNA 손상더 손상 DNA보다 여행 작은 DNA 조각을 만듭니다. 혜성 분석, 이미지 분석 소프트웨어, 조치하고 핵에서 마이그레이션 DNA와 핵에서 DNA의 전체 형광 강도를 비교합니다. 마이그레이션 DNA의 형광 신호가 DNA 손상에 비례합니다. 긴 밝은 DNA 꼬리 증가 DNA 손상을 의미합니다. 측정 매개 변수 중 일부는 꼬리에있는 DNA의 금액과 꼬리, 꼬리 길이 DNA의 유통 및 DNA의 비율 모두의 척도입니다 꼬리 순간입니다. 이 분석은 DNA 손상의 해상도가 복구가 자리를 차지하게 때문에뿐만 아니라 DNA 복구를 측정 할 수 있습니다. 감도의 제한 diploid 포유류의 세포 1,2 당 약 50 스트랜드 나누기입니다. 같은 etoposide와 같은 DNA 손상 대리인과 치료 세포는 긍정적 인 컨트롤로 사용할 수 있습니다. 따라서 혜성 분석은 DNA 손상을 측정 할 수있는 신속하고 효과적인 절차입니다.

Watch this Scientific Journal Video about Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay at JoVE.com

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

혜성 분석은 알칼리 불안정한 사이트와 hippocampal 뉴런을 포함한 모든 세포의 DNA에 DNA-DNA/DNA-protein 상호 링크 등 단일 및 이중 가닥 휴식을 검출 효율적인 방법입니다. 방법은 DNA 손상의 양의 차이로 인해 전기 분야에서 DNA의 차동 마이그레이션을 활용합니다.

혜성 분석을 사용하여 Hippocampal 뉴런의 DNA 손상을 시금

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

assaying-dna-damage-in-hippocampal-neurons-using-the-comet-assay

Research

JoVE Journal

Neuroscience

27.1K 조회수.  University of Alabama-Birmingham. 혜성 분석은 알칼리 불안정한 사이트와 hippocampal 뉴런을 포함한 모든 세포의 DNA에 DNA-DNA/DNA-protein 상호 링크 등 단일 및 이중 가닥 휴식을 검출 효율적인 방법입니다. 방법은 DNA 손상의 양의 차이로 인해 전기 분야에서 DNA의 차동 마이그레이션을 활용합니다.

비디오: Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &amp;#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

유전자 변형을 사용하여 시금 β - 아밀로이드 독성 C. 엘레간스 모델

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

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신경과학

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Neuromodulation 및 Mitochondrial 교통 : 긴 기간이 이상 Hippocampal 뉴런의 라이브 영상

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

태아 쥐의 Hippocampal 신경 세포의 분리 및 문화

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

차 해마 신경 세포의 칼슘 인산염 형질

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

구조적 조명 현미경을 사용하여 쥐 차 해마 신경 세포의 돌기 쪽 이미징

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

하는 세포 칼슘의 형광 및 생물 발광 영상<sup&gt; 2 +</sup&gt; 노인 해마 신경 세포에서

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

스트라이프 분석은 해리 해마 신경 세포를 사용하여 단백질 기판의 매력적인 또는 혐오감 활동을 연구하는

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

골지 - 콕스 방법을 이용한 고령의 해마 상피 세포의 수지상 돌기 복잡성 평가

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

교양된 Hippocampal 신경에 시 냅 스 소포 Endocytosis를 측정

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

성인 해마 신경 전구세포의 회로 특정 조절 을 시술

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate ...