Bartly J. Mondino MD, 줄스 스타 인 안구 연구소, UCLA의 이사;, 그리고 라미 네즈 - 솔리스, 재키 선배 화이트와 Sanger 연구소, 웰컴 트러스트 게놈 캠퍼스에서 쟌 Estabel 실명 방지를위한 연구. 이 연구는 안과 및 Visual 연구에서 동물의 사용에 대한 ARVO 보호 정책을 준수합니다.

1. 무딘 절개 : 고정되지 않은 표본에서 마우스 눈의 핵의 제거 <ol><li> 노출과 뒤쪽 세계 (안구) 표면에 대한 액세스를 개선하기 위해 눈꺼풀를 분열. </li><li> 궤도의 (이하) 세계 (눈 소켓) 뒤에 곡선 드레스 forcep를 놓습니다. Mahajan Sharptip 드레싱 forcep은 (재료 및 시약 표 참조)이 단계를 쉽게하기 위해 뾰족한 팁을 사용자 정의 악기입니다. </li><li> forcep을 닫고 세계를 쥐어 피하려면 조심하면서 전 세계 뒤에 궤도 결합 조직과 시신경을 파악. </li><li> 부드럽게 위쪽으로 forcep을 뽑아 궤도에서 눈알을 뽑아 내다. 흰색 스레드와 같은 조직은 시신경입니다. </li><li> PBS의 눈을 놓으십시오. 30 게이지 바늘을 사용하여, 하나의 구멍이 안구에 바늘 1~2밀리미터를 삽입하여 limbus에 바로 뒤쪽 상처를합니다. 이 안구 조직을 침투하기가 달리없는 등 글 루타 알데히드 등의 fixatives는,,, 실내 직접 수행 할 수 있습니다적이있는 눈. 이 정착액 등 paraformaldehyde 또는 알코올 등, 관심 안구 조직에 diffusing 할 경우이 찔린 상처를 만들 필요하지 않을 수 있습니다. </li><li> 즉시 침지 고정 용 정착액으로 전체 눈을 놓습니다. </li></ol> 2. 샤프 해부 : 마우스 눈 다음 재관류 고정의 핵의 제거 <ol><li> 거리에 세계에서 눈꺼풀을 보유하는 곡선 colibri forcep을 사용합니다. </li><li> 전 세계에 평행 동양 곡선 Westcott의 분해 가위, 궤도의 뒷면을 향해 목표로하는 동안. </li><li> 하부, 중간, 우수한, 그리고 측면 측면에서 세계를 둘러싸고있는 고정 결합 조직을 잘라. </li><li> 세계 뒤에 곡선 forcep을 놓고 세계에 눌러없이 결합 조직을 파악하고, 눈을 명백히하다 앞으로 당긴다. 궤도 결합 조직은 관류 고정에서 치열한이기 때문에, 궤도 조직의 추가 절단 완전히 글로브를 공개 할 필요가있을 수 있습니다5. </li></ol> 3. 고정 및 패키징 고정 조직의 배송은 적절한 라벨 및 포장을 포함하여 기관 및 우편 서비스 요구 사항을 준수해야합니다. 다음 프로토콜은 주위 온도에서 비 전염성 생물 샘플을 출하의 예입니다. 이 3 포장 층 있지만 클래스 A / B 생물학적 물질 또는 액체 질소 라벨이 필요합니다. <ol><li> 고정 짧은 경우 최소 3-ML의 정착액, 포스트 정착액 버퍼로 5 ML 유리 섬광 유리 병에 눈을 넣습니다. 유리 병의 뚜껑에있는 추적 번호를 작성하고 Parafilm로 밀봉. </li><li> 생물학적 샘플로 장소 섬광 튜브는 배송 컨테이너를 승인했습니다. 샘플이 배치되는 밀폐 용기, 흡수 중간 계층 및 보호 외부 층 : 우리 연구소에서 예를 들어, 승인 컨테이너는 3 층이 필요합니다. </li><li> 버블 랩 패키지로 한 다음 응용 프로그램에 컨테이너를 놓고적절한 서류와 함께 roved 실험실 배송 컨테이너. </li></ol> 4. 대표 결과 눈의 조직 학적 시험은 Hematoxylin과 Eosin의 착색, 효소 표현 감지, 전송 전자 현미경, 그리고 immunohistochemistry 등의 다양한 방법으로 수행 할 수 있습니다. 4 % paraformaldehyde에 침적 고정 후, 조직은 파라핀과 마이크로톰에 sectioned에 포함되었다. 높은 처리량 phenotyping, 우리는 학생 - 시신경 부분을 분석이 눈에 샘플 모든 조직 유형 (그림 1). 조직의 제는 lacZ 표현 (그림 2), 세포 세포 소기관의 전송 전자 현미경 (그림 3) 및 항체 (그림 4)와 특정 분자의 표현을 검토했다. 우리의 표현형 검사 방법에서는 (latera 코에 대해 (중간 안각)과 측두엽과 눈의 방향을 유지하기 위해 중요하지리터 안각)면. 눈의 방향이 필요한 경우 최소한 두 가지 옵션이 있습니다. 핵의 제거 후, ​​우리는 휴대용 소작 장치와 세시 위치에 시간적 각막에 빛 소작을 적용했습니다. 병변은 조직 학적 소견에 분명하고 조직 학적 잉크와 같은 처리하는 동안 손실되지 않습니다. 단점은 그 소작 손해 시험을위한 중요한 조직이 될 수있는 각막이다. 다른 옵션은 extraocular 근육 삽입을 식별 할 수있는 숙련 된 해부학자가 필요합니다. stereomicroscope에서 하부 우수한 경사 근육 삽입의 식별이 세계의 하부 및 뛰어난 및 측면을 표시합니다. 3 중 오리엔테이션 방법으로는 오른쪽 또는 왼쪽 눈으로 표본을 추적하는 것이 중요합니다. 함께이 sectioning하기 전에 정확한 전 방향을 할 수 있습니다. <img src="/files/ftp_upload/3184/3184fig1.jpg" alt="그림 1" /> 1 그림. 마우스 눈 조직 학적 이미지는 afte<html> R 원격 획득. 마우스 눈은 4 % paraformaldehyde로 관류 고정 후 날카로운 절개로 enucleated했다. Hematoxylin과 Eosin 물들 A.의 학생 - 시신경이 섹션은 모든 조직 substructures의 보존을 보여줍니다. 시신경이 없으면, ON. , IPL, 내부 plexiform 레이어, INL, 내부 핵 계층, OPL, 외부 plexiform 레이어, ONL, 외부 RGC, 망막 신경절 세포 : 일반 망막의 (녹색 규모의 바 = 500 마이크론) B. 높은 배율의 전망은 판상 구조를 보여줍니다 핵 레이어 (photoreceptor 세포) (화살표), OS, photoreceptor 외부 세그먼트; RPE, 망막 안료 상피, C, 맥락막, photoreceptor 내부 세그먼트 (화살표 머리)입니다. (녹색 규모 바 = 50 마이크론) C.는 정상적인 망막에 비해,이 표본은 photoreceptor 변성에 얇은 때문입니다. ONL는 있으며, OS는 (화살표) 완전히 결석입니다. (녹색 규모 줄이 = 50 마이크론) &quot;ALT =&quot;그림 2 &quot;/&gt; 유전자 변형 쥐 그림 2. lacZ의 표현. 눈 (5 MM K3Fe (CN) 6, 5 MM K4Fe (CN) 6, 2 MM MgCl2, 0.02 % NP40 무딘 절개로 enucleated 1 MG / ML X-여자 솔루션에 몰두했다 37의 밤 및 0.1 % 나트륨 deoxycholate) ° C. 눈은 30 분 마이크로톰에 sectioned 2 % formaldehyde/0.2 %의 글 루타 알데히드에서 수정되었습니다. X-여자 제품 (파란색) 레이블 : A. Ciliary 상피, B. 각막 상피와 C. 망막의 망막 신경절 세포 (RGC) 계층. INL, 내부 핵 계층, ONL, 바깥 쪽 핵 층. <img src="/files/ftp_upload/3184/3184fig3.jpg" alt="그림 3" /> 그림 3. 망막 색소 상피 (RPE)의 전송 전자 현미경. 찔린 상처의 무딘 절개과 창조 후, 눈을 집중했다는 0.1M 나트륨 인산염 버퍼에 2.5 %의 Paraformaldehyde / 2.5 %의 글 루타 알데히드에 고정. 눈이 secon했다darily, 1 % 오스뮴의 textroxide에서 수정 서서히 건조하고, Spurr의 수지에 포함. 조직은 90 나노 미터에서 sectioned이고 Formvar로 덮여 구리 슬롯 격자에 배치하고 전송 전자 현미경을 사용하여 이미징. 현미경은 photoreceptor 외부 neurosensory 망막의 세그먼트, 망막 안료 상피 내 melanosomes (M), 그리고 Bruch의 멤브레인을 보여줍니다. <img src="/files/ftp_upload/3184/3184fig4.jpg" alt="그림 4" /> 4 그림. 마우스 유리체에서 초과 산화물 dismutase-3 Immunohistochemical 라벨 (녹색). 눈이 무딘 절개로 enucleated와 4 % paraformaldehyde에 침적 고정을 받았습니다되었습니다. 그들은 마이크로톰을 사용하여 파라핀과 sectioned에 포함되었습니다. 1시 50분을 희석 조직 섹션은 토끼 polyclonal SOD3 항혈청과 incubated되었습니다. SOD3 표현은 염소 안티 - 토끼 IgG (H + L) - 알렉사 형석 488 복합 차 항체를 사용하여 감지되었습니다. SOD3의 라벨은 GRE에 표시됩니다ko 페이지를 참조하십시오.

<ol>
	<li>Song, B. J., Tsang, S. H., Lin, C. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+models+of+retinal+degeneration+and+targets+for+gene+therapy.">Genetic models of retinal degeneration and targets for gene therapy.</a> Gene Ther. Mol. Biol. 11, 229-262 (2007).</li><li>Mahajan, V. B., Mondino, B. J., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+Mouse+Eye+Phenomics+System.">A high-throughput Mouse Eye Phenomics System.</a> Cold Spring Harbor Laboratories, Mouse Genetics Meeting. , (2010).</li><li>Smith, R. S., John, S. W. M., Nishina, P. M., Sundberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In Research Methods For Mutant Mice. , (2002).</li><li>Chang, B., Hawes, N. L., Hurd, R. E., Davisson, M. T., Nusinowitz, S., Heckenlively, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+mutants+in+the+mouse.">Retinal degeneration mutants in the mouse.</a> Vision Res. 42, 517-525 (2002).</li><li>Anderson, M. G., Smith, R. S., Hawes, N. L., Zabaleta, A., Chang, B., Wiggs, J. L., John, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+genes+encoding+melanosomal+proteins+cause+pigmentary+glaucoma+in+DBA/2J+mice.">Mutations in genes encoding melanosomal proteins cause pigmentary glaucoma in DBA/2J mice.</a> Nat. Genet. 30, 81-85 (2002).</li><li>Hawes, N. L., Smith, R. S., Chang, B., Davisson, M., Heckenlively, J. R., John, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+fundus+photography+and+angiography:+a+catalogue+of+normal+and+mutant+phenotypes.">Mouse fundus photography and angiography: a catalogue of normal and mutant phenotypes.</a> Mol. Vis. 5, 22-22 (1999).</li><li>Won, J., Shi, L. Y., Hicks, W., Wang, J., Hurd, R., Naggert, J. K., Chang, B., Nishina, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+resources+for+vision+research.">Mouse model resources for vision research.</a> J. Ophthalmol. 2011, 391384-391384 (2011).</li><li>Pinto, L. H., Vitaterna, M. H., Siepka, S. M., Shimomura, K., Lumayag, S., Baker, M., Fenner, D., Mullins, R. F., Sheffield, V. C., Stone, E. M., Heffron, E., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Results+from+screening+over+9000+mutation-bearing+mice+for+defects+in+the+electroretinogram+and+appearance+of+the+fundus.">Results from screening over 9000 mutation-bearing mice for defects in the electroretinogram and appearance of the fundus.</a> Vision. Res. 44, 3335-3345 (2004).</li><li>Heckenlively, J. R., Winston, J. V., Roderick, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+mouse+retinal+degenerations.+I.+Correlation+of+indirect+ophthalmoscopy,+electroretinograms,+and+histology.">Screening for mouse retinal degenerations. I. Correlation of indirect ophthalmoscopy, electroretinograms, and histology.</a> Doc. Ophthalmol. 71, 229-239 (1989).</li><li>Tsang, S. H., Gouras, P., Yamashita, C. K., Kjeldbye, H., Fisher, J., Farber, D. B., Goff, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+in+mice+lacking+the+gamma+subunit+of+the+rod+cGMP+phosphodiesterase.">Retinal degeneration in mice lacking the gamma subunit of the rod cGMP phosphodiesterase.</a> Science. 272, 1026-1029 (1996).</li><li>Tsang, S. H., Woodruff, M. L., Jun, L., Mahajan, V., Yamashita, C. K., Pedersen, R., Lin, C. S., Goff, S. P., Rosenberg, T., Larsen, M., Farber, D. B., Nusinowitz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+carrying+the+H258N+mutation+in+the+gene+encoding+the+beta-subunit+of+phosphodiesterase-6+(PDE6B)+provide+a+model+for+human+congenital+stationary+night+blindness.">Transgenic mice carrying the H258N mutation in the gene encoding the beta-subunit of phosphodiesterase-6 (PDE6B) provide a model for human congenital stationary night blindness.</a> Hum. Mutat. 28, 243-254 (2007).</li></ol>

관심 없음 충돌이 선언 없습니다.

대부분의 유전자 변형 마우스는 눈을 검사하지 않는 실험실에 존재합니다. 우리 비디오 기술은 눈 작은 경험이있는 실험실에서 조직 획득을 최적화 할 수 원격 수술 기술 이전을위한 간단한 표준화 된 방법을 보여줍니다. 이 비디오 기술은 의미있는 비교 phenotypic와 분자 연구를 방지 비 표준화 조직 수집 및 고정 방법에 의한 전문 사이트 제한된 수의를 사용하는 것입니다 높은 처리량 phenotyping에...

<table border="1"><tbody><tr><th> 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 코멘트 </th></tr><tr><td> 구부러진 드레싱 Forcep </td><td> Storz Ophthalmics </td><td> E1408 </td><td></td></tr><tr><td> Mahajan Sharptip 드레싱 forcep </td><td> Storz Ophthalmics </td><td> E1406 (REF SP7-64520) </td><td></td></tr><tr><td> 곡선 Westcott의 가위 </td><td> Storz Ophthalmics </td><td> E3321 WH </td><td></td></tr><tr><td> 15 ° BD 비버 Microsurgical 블레이드 </td><td> Becton - 디킨슨 </td><td> 374881 </td><td></td></tr><tr><td> 0.22 미세 Castroviejo Suturing의 포셉 </td><td> Storz Ophthalmics </td><td> E1805 </td><td></td></tr><tr><td> 0.12 Colibri 포셉 </td><td> Storz Ophthalmics </td><td> 132분의 2 </td><td></td></tr><tr><td> 30 게이지 바늘 </td><td> Becton - 디킨슨 </td><td> 305128 </td><td></td></tr><tr><td> 바이오 해저드 메일 서버 </td><td> 어부 </td><td> 03-523-4 </td><td></td></tr><tr><td> Parafilm </td><td> 어부 </td><td> 13-374-10 </td><td></td></tr><tr><td> 유리 섬광 튜브 </td><td> 휘튼 </td><td> 4500413033 </td><td></td></tr><tr><td> PBS, pH를 7.4 </td><td> Invitrogen </td><td> 70011-044 </td><td></td></tr><tr><td> 16 % Paraformaldehyde </td><td> 전자 현미경 과학 </td><td> 15,700 </td><td></td></tr><tr><td> 0.1M 나트륨 인산염 버퍼에 2.5 %의 Paraformaldehyde / 2.5 %의 글 루타 알데히드 </td><td> 전자 현미경 과학 </td><td> 15700 및 16300 </td><td> 실험실에서 혼합 </td></tr><tr><td> 50 % 글 루타 알데히드 </td><td> 전자 현미경 과학 </td><td> 16,300 </td><td></td></tr><tr><td> 0.25 % Formvar </td><td> 전자 현미경 과학 </td><td> 15,810 </td><td></td></tr><tr><td> 순경슬롯 그리드 당 </td><td> 전자 현미경 과학 </td><td> M2010-CR </td><td></td></tr><tr><td> 4 % 오스뮴의 Tetroxide </td><td> 전자 현미경 과학 </td><td> 19,140 </td><td></td></tr><tr><td> 안티 SOD3 항체 </td><td> Abcam </td><td> Ab21974 </td><td></td></tr><tr><td> 염소 안티 - 토끼 알렉사 형석 488 </td><td> Invitrogen </td><td> A11070 </td><td></td></tr><tr><td> Spurr의 삽입 수지 </td><td> 전자 현미경 과학 </td><td> 14,300 </td><td></td></tr></tbody></table>

mouse eye enucleation for remote high-throughput phenotyping

마우스 눈은 인간의 안과 질환의 병진 연구의 중요한 유전 모델입니다. 이러한 황반 변성, photoreceptor 변성, 백내장, 녹내장, retinoblastoma, 그리고 당뇨병 성 망막증과 같은 인간에 실명을 야기하는 안구 질환을 치료는 1-5 대부분의 유전자 변형과 녹아웃 마우스가 아닌 안과 질환을 연구하기 위해 실험실에 의해 생성되었습니다. 유전자 변형 마우스에 recapitulated하지만, 한 장기 시스템 사이의 유전 절약은 동일한 유전자의 많은도 안구 개발과 질병에 역할을 할 수 있습니다 제안합니다. 따라서,이 마우스는 눈에 새로운 유전자형 - 표현형의 상관 관계를 발견하기위한 중요한 자원을 나타냅니다. 이 마우스는 전 세계에 흩어져 있기 때문에, 취득, 유지 관리 및 효율적 비용 효율적인 방법으로 표현형을하기가 어렵습니다. 따라서, 대부분의 높은 처리량 안과 phenotyping 화면은 라이브 쥐에 눈을 검토하기 위해 안과 전문 현장에서 필요로 몇 가지 위치로 제한됩니다. 6-9는 우리 연구실에서 개발 한 대체 접근 방식은 유전자 변형 마우스 눈 크고 작은 규모의 설문 조사에서 사용할 수있는 원격 조직 - 취득하는 방법입니다. 비디오 기반의 수술 기술 이전, 조직 고정 및 배송을위한 표준화 된 절차는 실험실 돌연변이 동물로부터 전체 눈을 수집하고 분자 및 형태학 phenotyping에 보내 할 수 있습니다. 이 비디오 문서에서는, 우리는 원격 phenotyping 분석을 위해 떼어 낸 및 재관류 모두 고정 마우스 눈을 명백히하다하고 전송하는 기술을 제시한다.

Watch this Scientific Journal Video about Mouse Eye Enucleation for Remote High-throughput Phenotyping at JoVE.com

Mouse Eye Enucleation for Remote High-throughput Phenotyping

분해 기술은 높은 처리량 화면에서 phenotyping을 수행 할 조직 고정에 대한 마우스 눈의 핵의 제거를 보여줍니다.

원격 높은 처리량 Phenotyping에 마우스 눈 핵의 제거

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

mouse-eye-enucleation-for-remote-high-throughput-phenotyping

Research

JoVE Journal

Medicine

20.1K 조회수.  University of Iowa. 분해 기술은 높은 처리량 화면에서 phenotyping을 수행 할 조직 고정에 대한 마우스 눈의 핵의 제거를 보여줍니다.

비디오: Mouse Eye Enucleation for Remote High-throughput Phenotyping

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.
The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1, followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis 2-4. Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup.
This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

단백질 치료제 제조 셀 라인의 개발을위한 높은 처리량 자동 플랫폼

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

연구

의학

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

Angiogenesis 연구에 대한 각막 포켓 분석의 마우스 모델

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

의식 억제 마우스에 인슈린 과다 - euglycemic 클램프

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

Remote Magnetic Navigation for Accurate, Real-time Catheter Positioning and Ablation in Cardiac Electrophysiology Procedures

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac substrates such as left atrial flutter. This protocol describes all the clinical and invasive interventional steps performed during a human electrophysiological study and ablation to assess the accuracy, safety and real-time navigation of the Catheter Guidance, Control and Imaging (CGCI) system. Patients who underwent ablation of a right or left atrium flutter substrate were included. Specifically, data from three left atrial flutter and two counterclockwise right atrial flutter procedures are shown in this report. One representative left atrial flutter procedure is shown in the movie. This system is based on eight coil-core electromagnets, which generate a dynamic magnetic field focused on the heart. Remote navigation by rapid changes (msec) in the magnetic field magnitude and a very flexible magnetized catheter allow real-time closed-loop integration and accurate, stable positioning and ablation of the arrhythmogenic substrate.

This report provides a detailed description of a new remote navigation system based on magnetic driven forces, which has been recently introduced as a new robotic tool for human cardiac electrophysiology procedures.

심장 전기 생리학 절차의 정확한 실시간 카테 테르 위치와 절제 원격 자기 탐색

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac ...

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2R&alpha;5, TNFR16, HGF7, IL-88, elafin2, and REG3&alpha;3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

급성 이식 - 대 - 호스트 질병의 생체의 유효성을 검사에 대한 높은 처리량 순차적 엘리사

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

신경 보호 전략에 대한 치료 요인의 납품을위한 설계 성인 줄기 세포의 높은 처리량 특성

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

마우스 망막 순환에서 유동 역학을 연구하기위한 적혈구 및 백혈구의 형광 염료 표지

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate (HoLEP).

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the quality of life. BPH includes 2 major treatments: medication and surgery. Surgical treatment is often the most effective and final intervention. Holmium laser enucleation of the prostate (HoLEP), one of the most efficient surgical procedures for BPH, is conducted transurethrally. During surgery, the most important and most difficult part is to locate the surgical capsule of the prostate, which is consistent to the idea of anatomic surgery. Different skills may be needed in treating different parts of the prostate. HoLEP tends to be efficient and safe with good hemostatic properties, and able to treat bladder calculus, which may be the complications of BPH. The technique is particularly suitable for prostate of varying volumes and sizes. Indeed, HoLEP also presents certain disadvantages, such as a long learning curve and costly equipment. Regardless, this method may be the "new standard" and the excellent method for the surgical treatment of BPH.

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate HoLEP.

Here we present a safe and efficient protocol, holmium laser enucleation of the prostate, to treat benign prostatic hyperplasia.

양성 전립선 비 대 증에 대 한 수술 치료: Holmium 레이저 Enucleation (HoLEP) 전립선의.

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

마우스 모델 Rubeosis Iridis의 아이리스 Neovascularization 펑크 유발

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.