우리는 기꺼이 웨인 스테이트 유니버 시티 (MJA), 연구 (SMV)를 노화에 대한 미국 재단에서 부여하고 독립 직업 전환 의생명 이미징 국립 연구소에서 수상 (R00EB007129)과 국립 연구소의 생물로 패스에서 시작 자금을 인정 보건 (MJA).

1. LnCl 3 소금을 사용 Metalation <ol><li> 30-265 mm의 솔루션을 생산하기 위해 물속에 리간드를 해산. 리간드 2 - (4 - isothiocyanatobenzyl) diethylenetriamine pentaacetic의 산성 (P - SCN - BN - DTPA)는 73 mm의 농도에서이 비디오에 사용되었습니다. </li><li> 1 M NH 4 OH를 추가하여 5.5 및 7.0 사이의 리간드의 솔루션의 산도를 조정합니다. 이 비디오에서, 1 M NH 4 OH 솔루션의 0.2 ML 사용되었다. </li><li> 5,000에서 1,000 사이 mm의 농도와 솔루션을 생산하기 위해 물에 LnCl 세 1-2에 상응하는 디졸브. 이 비디오에서는 EuCl 3 GdCl 3 111 mm의 농도에 사용되었습니다. 금속의 초과는 종종 완료 metalation을 유도하고 결과적으로 정화 작업을 단순화하는 데 사용됩니다. </li><li> 교반하면서 리간드의 솔루션에 LnCl 3 솔루션을 추가합니다. </li><li> LnCl 3 추가 후, 0.2 M NH 4 OH를 추가하여 5.5 및 7.0 사이에 발생하는 반응 혼합물의 산도를 조정합니다. 0.2 M NH 4 OH 솔루션의 0.5 ML 총이 동영상에 사용되었습니다. 귀하의 리간드가 산성에 민감한 기능 그룹을 포함하는 경우이 단계에서 산도를 여러 번 조정합니다. 주의 - 솔루션도 기본되면 모든 기본 기능 민감한 그룹, isothiocyanate와 같은 활용을 위해 사용할 수 없게 표시됩니다. </li><li> 산도 측정을 통해 반응을 모니터링합니다. 산도 상수 남아 때 반응이 완료됩니다. </li></ol> 2. 육아 산도의 검사 결과 (이 동영상에 포함되어 있지만, 기본 기능에 민감한 그룹없이 리간드에 도움되지 않음) <ol><li> ≥ 11 산도를 조절하기 위해 반응 혼합물에 NH 4 OH를 집중 추가합니다. 이 단계는 불용성 수산화 같은 uncomplexed 금속을 촉진합니다. </li><li> 0.2 μm의 필터를 통해 뜨는을 필터링합니다. 반응 혼합물은 필터를 나막신 경우, centrifuging 사전 필터링 decanting 권장합니다. </li><li> 투석을 수행하지 않는 경우, 압력 감소 (회전 증발 또는 동결 건조 권장)에서 용매 제거합니다. </li><li> 무료 란탄족가 남아있을 경우 단계 2.1-2.3 반복 수 있습니다. </li></ol> 3. 투석 검사 결과 <ol><li> 추가 길이 (샘플 볼륨의 약 10 %) 떠나면서 샘플 볼륨을 개최 적절한 길이 (제조 업체의 지침을 따르십)에 투석 튜브 컷. 이 비디오에서는 100-500 달튼 분자량 컷 - 오프 (MWCO) 멤브레인 사용되었지만, 활용이 metalation하기 전에 수행하는 경우 큰 MWCO 튜빙을 적절하게 사용할 수 있습니다. 또한, 투석 카세트가 원하는 경우 투석 튜브에 대한 대안으로 사용할 수 있습니다. </li><li> 제조 업체의 지침에 따라 해당되는 경우, 주변 온도에서 15 분 물에 컷 투석 튜브를 만끽해보세요. </li><li> 물 (dialysate)와 투석 저수지 (1 L의 비커이 동영상에 사용되었습니다) 입력합니다. dialysate 볼륨은 약 100x 그 샘플의해야합니다. </li><li> 팔짱을 두 번 튜브 끝과 투석 폐쇄 클램프로 튜브의 접힌 부분을 확보. 그것이 투석 도중에 폐쇄 상태를 유지하기 위해 고무 밴드와 폐쇄의 끝을 넣어. </li><li> 0.2 μm의 필터를 통해 반응 혼합물을 필터와 튜브를 찢어하지 않도록주의되는 튜브의 오픈 엔드에 여과물를로드합니다. 튜브를 닫습니다 충분한 머리 공간을 남겨주십시오. </li><li> 두 번, 폐쇄와 함께 안전하고, 단계 3.4에서와 같이 고무 밴드로 폐쇄 랩 튜브의 나머지 열린 끝을 접으십시오. </li><li> 고무 밴드를 사용하여 투석 튜브의 한쪽 끝을에서 클램프에 공기가 들어있는 유리관을 연결합니다. 다른 클램프로 모래가 담긴 병을 연결합니다. 이러한 튜브는 튜브가 dialysate에 포장되어 유지되었는지 확인합니다. </li><li> dialysate를 포함 투석 저수지에 전체 튜브를 놓습니다. </li><li> 주위 온도에서 느린 속도 (없음 vortexing)에서 자기 저어 접시를 사용하여 dialysate을 저어. </li><li> 하루 코스 (이 동영상에 dialysate가 2.5, 6.5, 및 11.5 H로 변경되었습니다)을 통해 배 dialysate를 변경하고 (투석의 20-28 H 총) 투석은 밤새 계속하실 수 있습니다. </li><li> dialysate에서 투석 튜브를 제거하고 신중하게 샘플을 제거 한 폐쇄를 엽니다. 물로 배 투석 튜브를 세척하고 샘플로 세탁을 결합. </li><li> 감소 압력 하에서 물을 제거합니다. 프리즈 건조는이 동영상에서 사용됩니다. </li></ol> 4. 무료로 금속의 존재를 평가 <ol><li> 아세테이트 버퍼에있는 금속 복잡한 디졸브 (버퍼 준비 : 400 ML의 물 속에 초산 1.4 ML를 해산, 1 M NH 4 OH와 5.8으로 산도를 조정, 500 ML 총 볼륨을 생성하는 물을 추가) 및 xylenol을 추가 주황색 표시등 (산도 5.8 버퍼에서 16 μm의). 이 비디오에서는 단지 0.3 MG는 버퍼의 0.3 ML에 해산되었으며 지표 솔루션 3 ML이 추가되었습니다. </li><li>보라색에 노란색에서 표시기의 색상 변화의 관찰을 통해 무료로 금속의 존재를 감지합니다. </li><li> 원하는 경우, 무료 금속의 금액은 교정 곡선 1을 만들어 계량하실 수 있습니다. 또는 염료 arsenazo III는 xylenol 오렌지 2 대신 사용할 수 있습니다. 무료로 금속이 남아있을 경우 예제는 더 이상 이전 특성에 투석, 탈염 열, 또는 고성능 액체 크로마 토그래피 (HPLC)를 사용하여 정화되어야합니다. </li></ol> 5. 수상 조정 번호 (Q)의 결정 <ol><li> H 2 O에서 복잡한 (~ 1 ㎜)을 포함하는 III - EU의 솔루션과 D 2 O.에서 같은 농도의 다른 솔루션을 준비 이전 분석, D 2 O 솔루션은 잔류 H 2 O.을 제거하는 세 번 증발와 D 2 O에 용해되어야합니다 </li><li> 깨끗한 쿠베트에 물 솔루션을 추가하고, spectrofluorometer에 쿠베트를 놓습니다. </li><li> 각 (~ 395 nm의와 ~ 595 nm의, 각각)의 맥시멈을 결정하기 위해 여기와 방출 스캔을 수행합니다. </li><li> 여기와 단계 5.3 여기 및 발광 슬릿 너비 (5 NM), 플래시 개수 (100), 초기 지연 (0.01 MS), 최대 지연 (13 MS)에서 결정 방출 파장 다음 매개 변수를 사용하여 인광 시간 붕괴 실험을 수행 및 지연 증가 (0.1 MS). 이러한 조건은 대부분의 단지에 적합하지만, 최대 지연과 증가 값은 매우 긴 또는 극단적으로 짧은 붕괴 시간과 함께 증가 또는 종족에 대한 감소 수 있습니다. </li><li> 단계 5.1 준비 D 2 O 솔루션 단계 5.4를 반복합니다. </li><li> 5.4 및 5.5에서 얻어진 발광 - 붕괴의 데이터에서, 강도 대 시간의 자연 로그를 플롯. 이 라인의 경사가 붕괴 속도 (τ -1) (그림 2)입니다. 이 비디오에서 Microsoft Excel 2007 원시 데이터에서 자연 로그 플롯을 생성하는 데 사용되었다. 호록스 및 동료 (EQ 1) 3 개발한 방정식의 붕괴 속도를 사용합니다. 귀하의 리간드가 금속에 맞춰 조정 OH 또는 NH 그룹을 포함하는 경우, 다음 방정식이 사용하는 세 전에 수정해야합니다. </li></ol> EQ 1 : <img src="/files/ftp_upload/2844/2844eq1.jpg" alt="방정식 1" /> 6. Relaxivity 측정 <ol><li> T 1 (세로 완화 시간) 또는 T 2 (가로 휴식 시간) 휴식 시간 분석기에 원하는 응용 프로그램 모드를 선택합니다. </li><li> GD의 다른 농도 III 함유 수성 용매에서 복잡한을 포함 샘플 시리즈를 준비합니다. 이 비디오에서는, 물은 0.625, 1.25, 2.50, 5.00, 10.0의 용매 및 솔루션으로 사용되었다, 0 MM이 준비되었습니다. 기타 수성 용매 또는 버퍼를 사용하지만, 그것은 빈으로 솔벤트를 사용하는 것이 중요합니다 수 있습니다. 예제의 마지막 볼륨 사용되고있는 악기에만 적용됩니다. </li><li> 악기의 샘플을 장소와 그것이 악기 (37 ° C이 비디오에서)의 온도 평형 5 분 앉아 보자. </li><li> T 1 T 2 (T 1과 T 2 대표적인 곡선 그림 3에 표시됩니다)에 대한 원활한 지수 곡선을 얻기 위해 소프트웨어의 매개 변수를 조정하여 휴식 시간 (s의 단위)를 확인합니다. </li><li> 단계 6.3 및 빈 포함한 모든 샘플 6.4 반복합니다. </li><li> S -1 단위로 측정된 T 1 T 2 값의 역수를 계산합니다. </li><li> T 1 -1 또는 T GD III 농도 비교 2 -1 값 (mm의 단위) 플롯. 때문에 GD III -가 포함된 단지의 흡습 특성, 원자 흡수 분광 광도법 또는 유도 결합 플라즈마 질량 분광법을 사용하여 GD III의 농도를 확인합니다. 직선과 줄거리를 맞습니다. 대표 플롯은 그림 4에 표시됩니다. </li><li> 장착되어 라인의 경사가 relaxivity (T 1과 T 2 각각에 대한 R 1 R 2)이며, MM -1 S -1의 단위를했다. </li></ol> 7. 대표 결과 이 프로토콜에서 설명하는 단계에 대한 대표적인 데이터는 테이블과 인물 섹션에 포함되었습니다. 프로토콜에서 설명한 물 조정 번호와 relaxivity 특성화 이외에, 그것은 표준 화학 기술을 사용하여 최종 제품을 특성화하는 것이 중요합니다. 및 EU III -가 포함된 단지는 그림 5에 표시된 - 화합물의 신분은 질량 분광법 및 질량 스펙트럼 대표 GD III에 대한 진단 동위 원소 패턴을 보여주는을 사용하여 얻을 수있다. 또한, 비 - GD III에 대한 </s최대&gt; 란탄족 함유 단지, NMR 분광법은 제품의 식별에 사용될 수 있습니다. 복합 순도, HPLC 또는 원소 분석을 특성화 또는 둘 모두를 사용할 수 있습니다. <img src="/files/ftp_upload/2844/2844fig1.jpg" alt="그림 1" /> 그림 1 metalation 및 정화를위한 일반적인 제도 :. 제도 metalation와 다른 정화 경로를 선택하는 이유에 대한 일반적인 절차를 묘사. <img src="/files/ftp_upload/2844/2844fig2.jpg" alt="그림 2" /> 그림 2 - 발광 강도를 줄거리 :. 강도 비교 섹션에서 5 시간의 자연 로그의 대표 플롯. 물, D 2 O 솔루션에 대한 인수와 비슷한 곡선에서 생성된 라인의 슬로프는 EU의 물 조정 번호 III 함유 단지를 특성화 EQ 1 사용됩니다. <img src="/files/ftp_upload/2844/2844fig3.jpg" alt="그림 3" /> 그림 3 휴식 붕괴 시간 곡선 :. (왼쪽) T 1 (오른쪽) T이 취득 대표 데이터입니다. 이러한 곡선 모양의 편차가 신뢰할 데이터를 생산합니다. <img src="/files/ftp_upload/2844/2844fig4.jpg" alt="그림 4" /> 그림 4 Relaxivity 결정 :. GD III의 농도 비교 1 / T 1 대표 플롯. 장착되어 라인의 경사가 relaxivity이며 MM -1 S -1의 단위를했다. <img src="/files/ftp_upload/2844/2844fig5.jpg" alt="그림 5" /> 그림 5 질량 스펙트럼 :.위한 진단 동위 원소 패턴을 보여주는 대표적인 질량 스펙트럼 (왼쪽) GD III 함유 단지과 (오른쪽) 약속 III 함유 단지 있습니다. 검은 가우스 봉우리는 이론 동위 원소 분포를 나타내며 빨간색 라인은 실제 데이터입니다.

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paramagnetic+metal+complexes+as+water+proton+relaxation+agents+for+NMR+imaging:+theory+and+design.">Paramagnetic metal complexes as water proton relaxation agents for NMR imaging: theory and design.</a> Chem. Rev. 87, 901-927 (1987).</li><li>Yoo, B., Pagel, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+responsive+MRI+contrast+agents+for+molecular+imaging.">An overview of responsive MRI contrast agents for molecular imaging.</a> Front. Biosci. 13, 1733-1752 (2008).</li><li>Pandya, S., Yu, J., Parker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+emissive+europium+and+terbium+complexes+for+molecular+imaging+and+sensing.">Engineering emissive europium and terbium complexes for molecular imaging and sensing.</a> Dalton Trans. 23, 2757-2766 (2006).</li><li>Nwe, K., Xu, H., Regino, C. A. S., Bernardo, M., Ileva, L., Riffle, L., Wong, K. J., Brechbiel, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+in+the+preparation+of+dendrimer-based+bifunctional+diethylenetriaminepentaacetic+acid+MR+contrast+agent+derivatives.">A new approach in the preparation of dendrimer-based bifunctional diethylenetriaminepentaacetic acid MR contrast agent derivatives.</a> Bioconjugate Chem. 20, 1412-1418 (2009).</li><li>Nwe, K., Bernardo, M., Regino, C. A. S., Williams, M., Brechbiel, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+MRI+properties+between+derivatized+DTPA+and+DOTA+gadolinium-dendrimer+conjugates.">Comparison of MRI properties between derivatized DTPA and DOTA gadolinium-dendrimer conjugates.</a> Bioorg. Med. Chem. 18, 5925-5931 (2010).</li><li>Caravan, P., Das, B., Deng, Q., Dumas, S., Jacques, V., Koerner, S. K., Kolodziej, A., Looby, R. J., Sun, W. -. C., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lysine+walk+to+high+relaxivity+collagen-targeted+MRI+contrast+agents.">A lysine walk to high relaxivity collagen-targeted MRI contrast agents.</a> Chem. Commun. , 430-432 (2009).</li><li>Le&#243;n-Rodr&#237;guez, L. M. D., Kovacs, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synthesis+and+chelation+chemistry+of+DOTA-peptide+conjugates.">The synthesis and chelation chemistry of DOTA-peptide conjugates.</a> Bioconjugate Chem. 19, 391-402 (2008).</li><li>Boswell, C. A., Eck, P. K., Regino, C. A. S., Bernardo, M., Wong, K. J., Milenic, D. E., Choyke, P. L., Brechbiel, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+characterization,+and+biological+evaluation+of+integrin+&#945;V&#946;3-targeted+PAMAM+dendrimers.">Synthesis, characterization, and biological evaluation of integrin &#945;V&#946;3-targeted PAMAM dendrimers.</a> Mol. Pharm. 5, 527-539 (2008).</li></ol>

관심 없음 충돌 선언하지 않습니다.

란탄족 기반 대비 요원 4-14을 포함 출판물의 증가 감안할 때, 그것은 치료가, 준비 정화하고, 재현성과 유사한 결과를 보장하기 위해 제품을 특성화로 촬영하는 것이 중요합니다. 이 단지는 종종 정화하고 상자 성체의 자연과 bioconjugation 사용할 수있는 기능 그룹의 감도에 따라 유기 분자에 상대적 성격 도전 간주됩니다. 우리는 란탄족 단지의 합성, 정제 및 특성에 대한 일반적인 방법을 ?...

<table border="1"><tbody><tr><th> 시약 및 장비 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> EuCl 3 • 6H 2 O </td><td> 시그마 - 알드리치 </td><td> 203254 - 5G </td></tr><tr><td> P - SCN - BN - DTPA </td><td> Macrocyclics </td><td> B - 305 </td></tr><tr><td> 암모늄 수산화 </td><td> EMD </td><td> AX1303 - 3 </td></tr><tr><td> 스펙트럼 / 포어 바이오 셀룰로오스 에스터 (CE) 투석 막 - 500 D MWCO </td><td> 피셔 사이 언티픽 </td><td> 68-671-24 </td></tr><tr><td> Millipore IC Millex - LG 필터 유닛 </td><td> 피셔 사이 언티픽 </td><td> SLLG C13 NL </td></tr><tr><td> xylenol 오렌지 tetrasodium 소금 </td><td> 알파 Aesar </td><td> 41,379 </td></tr><tr><td> 초산 </td><td> Fluka </td><td> 49,199 </td></tr><tr><td> D 2 O </td><td> 캠브리지 동위 원소 연구소 주식 회사 </td><td> DLM - 4-25 </td></tr><tr><td> 정수기 </td><td> ELGA </td><td> Purelab 울트라 </td></tr><tr><td> 고성능 액체 크로마 토그래피 및 질량 분광법 </td><td> Shimadzu </td><td> LCMS - 2010EV </td></tr><tr><td> 휴식 시간 분석기 </td><td> Bruker </td><td> mq60 minispec </td></tr><tr><td> UV - 마주 분광 광도계 </td><td> 피셔 사이 언티픽 </td><td> 20-624-00092 </td></tr><tr><td> 동결 건조기 </td><td> 피셔 사이 언티픽 </td><td> 10-030-133 </td></tr><tr><td> 산도 측정기 </td><td> 한나 스트 루먼트 </td><td> HI 221 </td></tr><tr><td> spectrofluorometer </td><td> 호리바 조빈 이본 </td><td> Fluoromax - 4 </td></tr><tr><td> 매튜 먼로의 분자량 계산기 버전 6.46은 2009 년 10월 17일을 다운로드 </td><td> <a href="http://ncrr.pnl.gov/software/">http://ncrr.pnl.gov/software/</a> </td><td> 분자량 계산기 </td></tr></tbody></table>

preparation, purification, and characterization of lanthanide complexes for use as contrast agents for magnetic resonance imaging

Polyaminopolycarboxylate 기반 리간드는 일반적으로 란탄족 이온을 킬레이트하는 데 사용하고, 그 결과 단지는 자기 공명 영상 (MRI)에 대한 대비를 대리인으로 유용합니다. 그들이 기능을 빠르고, 높은 순도를 허용 그룹 및 아민 반응 활성 에스테르 및 isothiocyanate 그룹 또는 thiol 반응 maleimides 통해 macromolecules 및 biomolecules에 높은 항복 활용을 포함하기 때문에 많은 상용 리간드 특히 유용합니다. 이러한 리간드의 metalation가 bioconjugation 화학, metalation 절차에 미묘한 차이의 분야에서 일반적인 지식으로 간주되는 동안 금속 시작 재료를 선택할 때 고려되어야합니다. 또한, 정화 및 특성이 존재하고, 가장 효과적인 절차의 선택에 대한 여러 옵션을 부분적으로 시작 물질의 선택에 따라 달라집니다. 이러한 미묘한 차이는 종종 출판 프로토콜에서 무시됩니다. 여기, 우리의 목표는 metalation, 정화하고, MRI (그​​림 1)에 대한 대비 에이전트로 사용할 수 있습니다 란탄족 단지의 특성화를위한 일반적인 방법을 입증하는 것입니다. 우리는이 책자는 생명 의학 과학자 시작 자료 및 정화 방법의 선택을 완화하여 일반적으로 사용되는 반응을 자신의 레퍼토리에 란탄족 complexation 반응을 통합할 수있게 것으로 기대합니다.

Watch this Scientific Journal Video about Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging at JoVE.com

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

우리는 metalation, 정화 및 란탄족 단지의 특성을 보여줍니다. 여기에서 설명한 단지는 자기 공명 영상을 사용하여 이러한 분자의 추적 활성화 macromolecules하는 복합 수 있습니다.

자기 공명 영상 준비, 정화하고, 명암 요원 이용 란탄족 단지의 특성화

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

preparation-purification-characterization-lanthanide-complexes-for

Research

JoVE Journal

Biology

14.9K 조회수.  Wayne State University. 우리는 metalation, 정화 및 란탄족 단지의 특성을 보여줍니다. 여기에서 설명한 단지는 자기 공명 영상을 사용하여 이러한 분자의 추적 활성화 macromolecules하는 복합 수 있습니다.

비디오: Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

이런 산화물로 hESCs과 hMSCs을 라벨링하는 MR 영상과 생체내 추적 비침습에 대한 Nanoparticles

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

연구

생물학

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

광학 이미징과 비침습 감지를위한 형광 염료와 함께 줄기 세포를 라벨링

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

자기 공명 영상을위한 인간 배아 줄기 세포의 체외 라벨에

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

단계 대비 및 차등 간섭 대비 (DIC) 현미경

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

의 분리 및 정제 Drosophila</em마그네틱 비드별로 정렬> 주변 뉴런

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

모델 생물체 유전자로 사용하기 위해 Medaka의 태아의 Microinjection

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

단백질 정제에서 사용하기 위해 자동화된 소수성 상호 작용 크로마 토그래피 칼럼 선정

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Quantifying Mixing using Magnetic Resonance Imaging

Mixing is a unit operation that combines two or more components into a homogeneous mixture. This work involves mixing two viscous liquid streams using an in-line static mixer. The mixer is a split-and-recombine design that employs shear and extensional flow to increase the interfacial contact between the components. A prototype split-and-recombine (SAR) mixer was constructed by aligning a series of thin laser-cut Poly (methyl methacrylate) (PMMA) plates held in place in a PVC pipe. Mixing in this device is illustrated in the photograph in Fig. 1. Red dye was added to a portion of the test fluid and used as the minor component being mixed into the major (undyed) component. At the inlet of the mixer, the injected layer of tracer fluid is split into two layers as it flows through the mixing section. On each subsequent mixing section, the number of horizontal layers is duplicated. Ultimately, the single stream of dye is uniformly dispersed throughout the cross section of the device.
 Using a non-Newtonian test fluid of 0.2% Carbopol and a doped tracer fluid of similar composition, mixing in the unit is visualized using magnetic resonance imaging (MRI). MRI is a very powerful experimental probe of molecular chemical and physical environment as well as sample structure on the length scales from microns to centimeters. This sensitivity has resulted in broad application of these techniques to characterize physical, chemical and/or biological properties of materials ranging from humans to foods to porous media 1, 2. The equipment and conditions used here are suitable for imaging liquids containing substantial amounts of NMR mobile 1H such as ordinary water and organic liquids including oils. Traditionally MRI has utilized super conducting magnets which are not suitable for industrial environments and not portable within a laboratory (Fig. 2). Recent advances in magnet technology have permitted the construction of large volume industrially compatible magnets suitable for imaging process flows. Here, MRI provides spatially resolved component concentrations at different axial locations during the mixing process. This work documents real-time mixing of highly viscous fluids via distributive mixing with an application to personal care products.

Magnetic resonance imaging (MRI) provides a powerful tool to evaluate the effectiveness of process equipment during operation. We discuss the use of MRI to visualize mixing in a static mixer. The application is relevant to personal care products, but can be applied to a broad range of food, chemical, biomass and biological fluids.

자기 공명 영상을 사용하여 혼합 Quantifying

Mixing is a unit operation that combines two or more components into a homogeneous mixture.  This work involves mixing two viscous liquid streams using an ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

이미징 및 면역 조직 화학에 대한 망막 Wholemount 준비를 위해 최적화 된 프로토콜

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

세대와 인간의 INO80 염색질 리모델링 단지 및 서브 콤플렉스의 정화

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...