Name: measuring the confluence of ipscs using an automated imaging system
Uploaded: 2020-06-10T13:30:00.000+00:00
Duration: 11 min 39 s
Description: Watch this Scientific Journal Video about Measuring the Confluence of iPSCs Using an Automated Imaging System at JoVE.com

이 연구는 Fondazione Bambino Gesù와 Ricerca Corrente (이탈리아 보건부)의 C.C. 보조금으로 지원되었습니다.  엔리코 베르티니 박사(신경과학과, 신경근 및 신경퇴행성 질환 부서, 밤비노 제수 어린이 연구 병원 분자 의학 연구실), 스테파니아 페트리니 박사(공초점 현미경 핵심 시설, 연구 실험실, 밤비노 제수 어린이 연구 병원), 줄리아 페리콜리(종양혈액학과, 유전자 및 세포 치료과, 밤비노 제수 어린이 연구 병원) Roberta Ferretti(종양 혈액학, 유전자 및 세포 치료학과, 어린이 연구 병원 밤비노 제수)는 과학적 토론과 기술적 도움을 제공합니다. 마리아 빈치는 "Children with Cancer UK 펠로우십"을 수상했습니다.

1. 96웰 플레이트 코팅
참고: 동일한 플레이트에서 다른 코팅을 테스트했지만 별도의 웰에서 테스트했습니다( 보충 파일 참조).
<ol><li>매트리겔을 DMEM에서 1:100으로 희석한다. 96웰 플레이트에 웰당 100μL를 추가하고 실온에서 1시간 동안 배양합니다. 그런 다음 용액을 제거하고 100μL의 DMEM으로 웰을 두 번 세척합니다.</li>
	<li>PBS (칼슘 및 마그네슘 포함)에 희석 된 라미닌 (20 μg / mL, LN-521). 웰에 100 μL를 첨가하고 4°C에서 밤새 배양한다. 다음날 세포를 파종하기 전에 DMEM으로 두 번 세척하십시오.</li>
	<li>희석 완충액에 비트로넥틴(10μg/mL)을 희석합니다. 96웰 플레이트에 웰당 100μL를 추가하고 실온에서 1시간 동안 배양한다. 세포를 도금하기 전에 PBS (칼슘과 마그네슘이없는)로 우물을 씻으십시오.</li>
	<li>HU-피브로넥틴(30μg/mL)을ddH2O에 희석하고 웰에 100μL를 넣고 실온에서 45분 동안 배양합니다. 그런 다음 세포를 파종하기 전에 배지로 웰을 씻으십시오.</li>
</ol>2. 배양에서 iPSC의 유지 관리
참고: iPSC는 상업적으로 구입했습니다. iPSC는 건강한 인간 섬유아세포에서 파생되었으며 에피솜 기술을 사용하여 재프로그래밍되었습니다.
<ol><li>-80°C 냉동고 또는 액체 질소로부터, 동결보존된 iPSCs를 37°C 수조에서 해동시킨다. 생물학적 안전 캐비닛으로 옮기기 전에 세포가 들어있는 바이알을 70 % 에탄올로 청소하십시오.</li>
	<li>15mL 멸균 원뿔형 튜브에서 1000μL 피펫으로 5mL의 사전 예열된 세포 배양 배지(예: mTeSR1)에 세포 현탁액을 한 방울씩 추가합니다.</li>
	<li>실온(RT)에서 5분 동안 304 x g 의 셀을 원심분리합니다.</li>
	<li>배지를 제거하고 세포 펠릿을 4mL의 세포 배양 배지에 재현탁합니다.</li>
	<li>세포 현탁액을 6웰 플레이트(6웰 세포 배양 접시당5 iPSC 10개)의 2개 웰에 플레이팅하고, 여기서 마우스 배아 섬유아세포(MEF)가 이전에 플레이팅되었습니다. DMEM (10 % 태아 소 혈청, 1 % L- 글루타민 및 1 % 페니실린-스트렙토 마이신 함유)에서 2.4 x 104 / cm2 의 밀도로 iPSC를 도금하기 이틀 전에 MEF를 시드합니다.</li>
	<li>파종 후 세포 배지에 10μM의 ROCK 억제제 Y-27632를 보충합니다.</li>
	<li>처음 4-5주 동안 MEF에서 iPSC를 성장시킨 다음 mTeSR1에서 관심 있는 코팅 중 하나(1단계 및 표 1 참조)를 사용하여 피더 프리 조건(MEF 프리 조건)에서 성장시킵니다.</li>
	<li>iPSC가 70-80% 합류점일 때 RT에서 3-5분 동안 0.5mM EDTA 처리를 사용하여 1:4 통과합니다. 6웰 플레이트의 경우 0.5mM EDTA 1mL를 추가합니다(또는 다른 유형의 플레이트의 경우 비례 양). 피더가없는 조건에서 새 웰로 옮기고 37 ° C, 5 % CO2, 20 % O2에서 배양합니다.</li>
	<li>매일 새로운 mTeSR1로 배지를 교체하고 2일마다 세포를 분할합니다.</li>
</ol>3. 세포 합류의 특성 분석
<ol><li>실험을 위해 96웰 플레이트를 사용하십시오.</li>
	<li>MEF가 계대배양되지 않았는지 확인하기 위해 피더가 없는 상태에서 최소 1개월 배양한 후 웰당 10,000개의 세포를 시드합니다. 일회용 계수 슬라이드를 사용하여 광학 현미경으로 세포를 계수하십시오.</li>
	<li>실험을 세 번 수행하십시오. 따라서 3 개의 웰에서 각 파종 조건을 테스트하십시오.</li>
	<li>명시야 모드에서 세포분석기를 사용하여 파종 후 1일째부터 자동 이미지 획득을 수행합니다. 5일 동안 24시간마다 자동 이미지 획득을 수행합니다. 실험 파라미터에 대한 자세한 내용은 보충 파일을 참조하십시오.</li>
	<li>자동 대비 및 자동 노출을 사용하여 셀을 더 잘 시각화합니다.</li>
	<li>합류 분석에 대한 분석 설정( 보충 파일 참조)을 웰당 60%, 80% 및 100%의 마스크를 적용하여 웰 경계의 빛 굴절로 인한 초점 변화를 평가합니다. 위에서 언급한 다른 마스크 분석 설정을 사용하여 각 시점에서 세포 합류를 분석합니다.</li>
</ol>4. 통계 분석
<ol><li>정량적 결과를 평균(SEM)의 표준 오차± 평균으로 보고합니다.</li>
	<li>서로 다른 코팅 조건의 전반적인 차이를 비교하려면 동일한 표본을 사용하여 데이터를 얻고 스튜던트 쌍 표본 t-검정을 수행합니다. 0.05 미만의 P 값은 통계적으로 유의한 것으로 간주되며 보고된 모든 p-값은 양측입니다.</li>
</ol>5. 세포골격 마이크로필라멘트의 특성 분석
<ol><li>RT에서 10분 동안 PBS 중 4% 파라포름알데히드(4% PFA)로 세포를 고정한 후 PBS에서 2회 세척(총 10분)합니다.</li>
	<li>100 μL의 블로킹 용액 (PBS 중 5 % BSA, 0.1 % 트리톤으로 구성)을 RT에서 1 시간 동안 각 웰에 첨가하십시오.</li>
	<li>블로킹 용액을 제거하고 샘플을 PBS로 10분 동안 두 번 세척한다.</li>
	<li>샘플당 100μL의 팔로이딘-접합체 작업 용액을 추가하고 RT에서 1시간 동안 배양합니다.</li>
	<li>PBS로 세포를 두 번 세척합니다 (RT에서 10 분).</li>
	<li>Hoechst 33342로 핵을 염색하고 RT에서 10분 동안 PBS에서 1:10000으로 희석합니다.</li>
	<li>Hoechst 용액을 제거하고 매번 10분 동안 PBS로 세포를 두 번 세척합니다.</li>
	<li>샘플을 H2O로 세척하고 화학 후드 아래에서 건조시킵니다.</li>
	<li>100μL의 장착 매체(예: PBS:글리세롤, 1:1)를 추가하여 세포를 덮고 샘플의 형광을 보존합니다.</li>
	<li>백색광 레이저(WLL) 소스와 405nm 다이오드 레이저가 장착된 레이저 스캐닝 컨포칼 현미경으로 Ex/Em 493/517nm에서 세포를 관찰합니다. HC PLAPO 40x 오일 이멀젼 대물렌즈를 사용하여 순차적 컨포칼 이미지를 획득합니다. 검사된 모든 샘플에 대해 동일한 레이저 출력, 빔 스플리터, 필터 설정, 핀홀 직경 및 스캔 모드를 사용합니다.</li>
</ol>

<ol>
	<li>Masotti, A., 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=Aged+iPSCs+display+an+uncommon+mitochondrial+appearance+and+fail+to+undergo+in+vitro+neurogenesis.">Aged iPSCs display an uncommon mitochondrial appearance and fail to undergo in vitro neurogenesis.</a> Aging (Albany NY). 6 (12), 1094-1108 (2014).</li><li>Xu, C., 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=Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells.">Feeder-free growth of undifferentiated human embryonic stem cells.</a> Nature Biotechnology. 19, 971-974 (2001).</li><li>Kleinman, H. K., 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=Isolation+and+characterization+of+type+IV+procollagen,+laminin,+and+heparan+sulfate+proteoglycan+from+the+EHS+sarcoma.">Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma.</a> Biochemistry. 21 (24), 6188-6193 (1982).</li><li>Rodin, S., 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=Clonal+culturing+of+human+embryonic+stem+cells+on+laminin-521/E-cadherin+matrix+in+defined+and+xeno-free+environment.">Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment.</a> Nature Communications. 5, 3195 (2014).</li><li>Rodin, S., Antonsson, L., Hovatta, O., Tryggvason, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monolayer+culturing+and+cloning+of+human+pluripotent+stem+cells+on+laminin-521+based+matrices+under+xeno-free+and+chemically+defined+conditions.">Monolayer culturing and cloning of human pluripotent stem cells on laminin-521 based matrices under xeno-free and chemically defined conditions.</a> Nature Protocols. 9 (10), 2354-2368 (2014).</li><li>Laperle, A., 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=&#945;-5+Laminin+Synthesized+by+Human+Pluripotent+Stem+Cells+Promotes+Self-Renewal.">&#945;-5 Laminin Synthesized by Human Pluripotent Stem Cells Promotes Self-Renewal.</a> Stem Cell Reports. 5 (2), 195-206 (2015).</li><li>Albalushi, H., 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=Laminin+521+stabilizes+the+pluripotency+expression+pattern+of+human+embryonic+stem+cells+initially+derived+on+feeder+cells.">Laminin 521 stabilizes the pluripotency expression pattern of human embryonic stem cells initially derived on feeder cells.</a> Stem Cell International. 2018, 7127042 (2018).</li><li>Zhang, D., 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=Niche-derived+laminin-511+promotes+midbrain+dopaminergic+neuron+survival+and+differentiation+through+YAP.">Niche-derived laminin-511 promotes midbrain dopaminergic neuron survival and differentiation through YAP.</a> Science Signaling. 10 (493), (2017).</li><li>Lu, H. F., 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+defined+xeno-free+and+feeder-free+culture+system+for+the+derivation,+expansion+and+direct+differentiation+of+transgene-free+patient-specific+induced+pluripotent+stem+cells.">A defined xeno-free and feeder-free culture system for the derivation, expansion and direct differentiation of transgene-free patient-specific induced pluripotent stem cells.</a> Biomaterials. 35 (9), 2816-2826 (2015).</li><li>Miyazaki, T., Nakatsuji, N., Suemori, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+slow+cooling+cryopreservation+for+human+pluripotent+stem+cells.">Optimization of slow cooling cryopreservation for human pluripotent stem cells.</a> Genesis. 52 (1), 49-55 (2014).</li><li>Bergstr&#246;m, R., Str&#246;m, S., Holm, F., Feki, A., Hovatta, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xeno-free+culture+of+human+pluripotent+stem+cells.">Xeno-free culture of human pluripotent stem cells.</a> Methods in Molecular Biology. 767, 125-136 (2011).</li><li>Braam, S. R., 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=Recombinant+vitronectin+is+a+functionally+defined+substrate+that+supports+human+embryonic+stem+cell+self-renewal+via+alphavbeta5+integrin.">Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via alphavbeta5 integrin.</a> Stem Cells. 26 (9), 2257-2265 (2008).</li><li>Chen, G., 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=Chemically+defined+conditions+for+human+iPSC+derivation+and+culture.">Chemically defined conditions for human iPSC derivation and culture.</a> Nature Methods. 8 (5), 424-429 (2008).</li><li>Li, J., 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=Impact+of+vitronectin+concentration+and+surface+properties+on+the+stable+propagation+of+human+embryonic+stem+cells.">Impact of vitronectin concentration and surface properties on the stable propagation of human embryonic stem cells.</a> Biointerphases. 5 (3), 132-142 (2010).</li><li>Prowse, A. B., 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=Long-term+culture+of+human+embryonic+stem+cells+on+recombinant+vitronectin+in+ascorbate+free+media.">Long-term culture of human embryonic stem cells on recombinant vitronectin in ascorbate free media.</a> Biomaterials. 31 (32), 8281-8288 (2010).</li><li>Rowland, T. J., 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=Roles+of+integrins+in+human+induced+pluripotent+stem+cell+growth+on+Matrigel+and+vitronectin.">Roles of integrins in human induced pluripotent stem cell growth on Matrigel and vitronectin.</a> Stem Cells and Development. 19 (8), 1231-1240 (2010).</li><li>Ruoslahti, E., Engvall, E., Hayman, E. G., Spiro, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+studies+on+amniotic+fluid+and+plasma+fibronectins.">Comparative studies on amniotic fluid and plasma fibronectins.</a> Biochemistry. 193 (1), 295-299 (1981).</li><li>Ni, H., Li, A., Simonsen, N., Wilkins, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+activation+by+dithiothreitol+or+Mn2++induces+a+ligand-occupied+conformation+and+exposure+of+a+novel+NH2-terminal+regulatory+site+on+the+beta1+integrin+chain.">Integrin activation by dithiothreitol or Mn2+ induces a ligand-occupied conformation and exposure of a novel NH2-terminal regulatory site on the beta1 integrin chain.</a> Biological Chemistry. 273 (14), 7981-7987 (1998).</li><li>Seltana, A., Basora, N., Beaulieu, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+epithelial+wound+healing+assay+in+an+epithelial-mesenchymal+co-culture+system.">Intestinal epithelial wound healing assay in an epithelial-mesenchymal co-culture system.</a> Wound Repair &amp; Regeneration. 18 (1), 114-122 (2010).</li><li>Amit, M., Shariki, C., Margulets, V., Itskovitz-Eldor, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder+layer-and+serum-free+culture+of+human+embryonic+stem+cells.">Feeder layer-and serum-free culture of human embryonic stem cells.</a> Biology of Reproduction. 70 (3), 837-845 (2004).</li><li>Vaheri, A., Mosher, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+molecular+weight,+cell+surface-associated+glyco-protein+(fibronectin)+lost+in+malignant+transformation.">High molecular weight, cell surface-associated glyco-protein (fibronectin) lost in malignant transformation.</a> Biochimica et Biophysica Acta. 516 (1), 1-25 (1978).</li><li>Mao, Y., Schwarzbauer, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+fibrillogenesis,+a+cell-mediated+matrix+assembly+process.">Fibronectin fibrillogenesis, a cell-mediated matrix assembly process.</a> Matrix Biology. 24 (6), 389-399 (2005).</li><li>Polyak, K., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transitions+between+epithelial+and+mesenchymal+states:+acquisition+of+malignant+and+stem+cell+traits.">Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits.</a> Nature Reviews Cancer. 9 (4), 265-273 (2009).</li><li>Kadler, K. E., Hill, A., Canty-Laird, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+fibrillogenesis:+fibronectin,+integrins,+and+minor+collagens+as+organizers+and+nucleators.">Collagen fibrillogenesis: fibronectin, integrins, and minor collagens as organizers and nucleators.</a> Current Opinion in Cell Biology. 20 (5), 495-501 (2008).</li><li>Hunt, G. C., Schwarzbauer, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tightening+the+connections+between+cadherins+and+fibronectin+matrix.">Tightening the connections between cadherins and fibronectin matrix.</a> Developmental Cell. 16 (3), 327-328 (2009).</li><li>Villa-Diaz, L. G., Ross, A. M., Lahann, J., Krebsbach, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+The+evolution+of+human+pluripotent+stem+cell+culture:+from+feeder+cells+to+synthetic+coatings.">Concise review: The evolution of human pluripotent stem cell culture: from feeder cells to synthetic coatings.</a> Stem Cells. 31 (1), 1-7 (2013).</li><li>Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126 (4), 677-689 (2006).</li><li>Vigilante, A., 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=Identifying+Extrinsic+versus+Intrinsic+Drivers+of+Variation+in+Cell+Behavior+in+Human+iPSC+Lines+from+Healthy+Donors.">Identifying Extrinsic versus Intrinsic Drivers of Variation in Cell Behavior in Human iPSC Lines from Healthy Donors.</a> Cell Reports. 26 (8), 2078-2087 (2019).</li></ol>

저자는 공개 할 것이 없습니다.

질병 모델링 및 향후 약물 스크리닝에 iPSC를 사용하고 정밀 의학에 적용 할 수 있기 때문에 iPSC는 관련성이 높은 기술이되며 이러한 이유로 배아 줄기 세포의 생리적 상황과 더 유사한 체외 배양 조건을 명확하게 이해할 필요가 있다고 생각합니다. 이러한 맥락에서 우리는 세포가 건강하고 미분화 상태를 유지할 수 있는 조건을 이해하기 위해 야생형 iPSC를 사용하여 다양한 ECM 코팅을 테스트했습?...

유도 만능 줄기 세포 (iPSC)는 체세포에서 얻어지며 다른 세포 유형으로 분화 될 수 있습니다. 그들은 종종 질병 발병 기전을 모델링하거나 약물 스크리닝을 수행하는 시스템으로 사용되며 개인화 된 의학의 맥락에서 사용될 수있는 잠재력을 제공합니다. iPSC는 큰 잠재력을 가지고 있기 때문에 신뢰할 수 있는 모델 시스템으로 사용하기 위해 완전히 특성화하는 것이 중요합니다. 우리는 이전에 저산소 환경에서 iPSC를 성장시키는 것의 중요성을 보여주었습니다.이 세포는 해당 과정에 의존하고 호기성 환경은 산화 환원 불균형을 유발할 수 있습니다1. iPSC는 또한 다른 배양 조건, 특히 세포 외 환경에 취약합니다. 배양 조건의 최적화는 그들을 건강하고 증식시키는 핵심 문제입니다. 건강한 iPSC 배양은 일반적으로 특정 인간 장애 또는 세포 과정의 분자, 세포 및 기능적 특징을 이해하는 데 사용되는 모델의 종말점인 건강한 분화 세포로 이어질 것입니다.
이 연구에서는 간단한 프로토콜을 사용하여 별도의 웰에서 서로 다른 코팅 조건을 사용하여 iPSC의 합류를 테스트했습니다. iPSC가 제대로 부착되기 위해서는 쥐 배아 섬유아세포(MEF)의 영양층이 필요하지만 iPSC와 MEF가 공존하면 두 개의 세포 집단이 존재하기 때문에 RNA 또는 단백질 추출과 같은 분석을 수행하기가 어렵습니다. 피더 층을 피하기 위해 세포 외 기질 (ECM)에 속하는 다양한 단백질을 사용하여 천연 세포 틈새를 재현하고 피더가없는 iPSC 배양을 가졌습니다. 특히, Matrigel은 엥겔브레스-홀름-스웜(EHS) 마우스 육종에서 추출한 가용화된 기저막 제제로, 세포외 기질 단백질(즉, 라미닌, 콜라겐 IV, 헤파란 설페이트 프로테오글리칸, 엔탁틴/니도겐 및 성장 인자)이 풍부합니다2,3. 다른 사용 된 코팅 조건은 대신 ECM을 구축하는 데 관련성이 알려진 정제 된 단백질입니다 : 라미닌 -521은 배아의 내부 세포 덩어리에서 인간 다 능성 줄기 세포 (hPSC)에 의해 분비되는 것으로 알려져 있으며 출생 후 신체에서 가장 흔한 라미닌 중 하나입니다 4,5,6,7,8,9, 10,11; 비트로넥틴은 hPSC 12,13,14,15,16의 성장 및 분화를 지원하는 것으로 알려진 이종 무함유 세포 배양 매트릭스입니다. 피브로넥틴은 척추동물의 발달과 만능 상태의 배아 줄기세포의 부착 및 유지에 중요한 ECM 단백질이다 17,18,19,20,21,22,23,24,25. 다양한 코팅 조건을 사용할 수 있기 때문에 iPSC의 합류에 미치는 영향 측면에서 비교합니다.

<table><tbody><tr><td>10 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile</td><td>Corning</td><td>4488</td><td>Tool</td></tr><tr><td>15 mL high-clarity polypropylene (PP) conical centrifuge tubes</td><td>Falcon</td><td>352097</td><td>Tool</td></tr><tr><td>1x PBS (With Ca&lt;sup&gt;2+&lt;/sup&gt;; Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td>Thermofisher</td><td>14040133</td><td>Medium</td></tr><tr><td>1x PBS (without Ca&lt;sup&gt;2+&lt;/sup&gt;; Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td>Euroclone</td><td>ECB4004L</td><td>Medium</td></tr><tr><td>5 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile</td><td>Corning</td><td>4487</td><td>Tool</td></tr><tr><td>Cell culture microplate, 96 WELL, PS, F-Bottom</td><td>Greiner Bio One</td><td>655090</td><td>Support</td></tr><tr><td>Cell culture plate, 6 well</td><td>Costar</td><td>3516</td><td>Support</td></tr><tr><td>DMEM (Dulbecco&#039;s Modified Eagle&#039;s Medium- high glucose)</td><td>Sigma</td><td>D5671</td><td>Medium</td></tr><tr><td>EDTA</td><td>Sigma</td><td>ED4SS-500g</td><td>Reagent</td></tr><tr><td>Epi Episomal iPSC Reprogramming Kit</td><td>Invitrogen</td><td>A15960</td><td>Reagent</td></tr><tr><td>FAST - READ 102</td><td>Biosigma</td><td>BVS100</td><td>Tool</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>10270106</td><td>Medium</td></tr><tr><td>Fibronectin</td><td>Merck</td><td>FC010</td><td>Coating</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td>Reagent</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td>MILLIQ</td><td></td></tr><tr><td>Hoechst</td><td>Thermofisher</td><td>33342</td><td>Reagent</td></tr><tr><td>Laminin 521</td><td>Stem Cell Technologies</td><td>77003</td><td>Coating</td></tr><tr><td>L-Glutamine (200 mM)</td><td>Gibco</td><td>LS25030081</td><td>Reagent</td></tr><tr><td>Matrigel</td><td>Corning Matrigel hESC-Qualified Matrix</td><td>354277</td><td>Coating</td></tr><tr><td>Mouse embryonic fibroblasts (MEF)</td><td>Life Technologies</td><td>A24903</td><td>Coating</td></tr><tr><td>MTESR1 Medium</td><td>Stem Cell Technologies</td><td>85851</td><td>Medium</td></tr><tr><td>MTESR1 Supplement</td><td>Stem Cell Technologies</td><td>85852</td><td>Medium</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Gibco</td><td>15140122</td><td>Reagent</td></tr><tr><td>Phalloidin</td><td>Sigma</td><td>P1951</td><td>Reagent</td></tr><tr><td>Vitronectin</td><td>Stem Cell Technologies</td><td>7180</td><td>Coating</td></tr><tr><td>Y-27632</td><td>Sigma</td><td>Y0503</td><td>Reagent</td></tr></tbody></table>

measuring the confluence of ipscs using an automated imaging system

이 연구는 다양한 ECM 코팅 기판에서 성장하는 iPSC가 세포 합류에 어떤 영향을 미칠 수 있는지 이해하는 데 중점을 둡니다. 성장 섭동을 피하기 위해 단일 세포 현탁액의 세포를 계산할 필요 없이 실시간으로 iPSC 합류를 평가하는 프로토콜이 확립되었습니다. 고함량 이미지 분석 시스템을 사용하여 시간이 지남에 따라 4개의 서로 다른 ECM에서 iPCS 합류를 자동화된 방식으로 평가했습니다. 부착성 iPSC의 세포 합류를 평가하기 위해 다양한 분석 설정을 사용했으며 60, 80 또는 100% 마스크가 적용되었는지 여부에 관계없이 약간의 차이(라미닌의 경우 24시간 및 48시간)만 관찰되었습니다. 우리는 또한 라미닌이 Matrigel, vitronectin 및 fibronectin에 비해 가장 좋은 합류로 이어진다는 것을 보여줍니다.

이 연구에서는 다양한 코팅 조건에서 성장할 때 iPSC의 합류를 조사했습니다. 세포 분석기를 사용하여 5 일 만에 3 배로 쉽게 유익한 결과를 얻을 수있었습니다. iPSC는 플라스틱 용기에 거의 부착되지 않고 증식을 지원하기 위해 코팅이 필요하기 때문에 세포 배양의 건강을 나타내고 분화 가능성을 반영할 수 있으므로 인간 iPSC의 합류를 모니터링하기로 결정했습니다. 시험관 내 확장 후, 우리는 iPSC...

Watch this Scientific Journal Video about Measuring the Confluence of iPSCs Using an Automated Imaging System at JoVE.com

Measuring the Confluence of iPSCs Using an Automated Imaging System

이 프로토콜의 목표는 다양한 세포외 기질(ECM) 코팅 조건을 비교하여 차등 코팅이 유도만능줄기세포(iPSC)의 성장 속도에 어떤 영향을 미치는지 평가하는 것입니다. 특히, 우리는 iPSC 배양의 최적 성장을 얻기위한 조건을 설정하는 것을 목표로합니다.

자동 이미징 시스템을 사용한 iPSC의 합류점 측정.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

measuring-the-confluence-of-ipscs-using-an-automated-imaging-system

Research

JoVE Journal

Biology

2.0K 조회수.  Ospedale Pediatrico Bambino Gesù, IRCCS. 이 프로토콜의 목표는 다양한 세포외 기질(ECM) 코팅 조건을 비교하여 차등 코팅이 유도만능줄기세포(iPSC)의 성장 속도에 어떤 영향을 미치는지 평가하는 것입니다. 특히, 우리는 iPSC 배양의 최적 성장을 얻기위한 조건을 설정하는 것을 목표로합니다.

비디오: Measuring the Confluence of iPSCs Using an Automated Imaging System

A Practical Guide on Coupling a Scanning Mobility Sizer and Inductively Coupled Plasma Mass Spectrometer (SMPS-ICPMS)

A large variety of analytical methods are available to characterize particles in aerosols and suspensions. The choice of the appropriate technique depends on the properties to be determined. In many fields information about particle size and chemical composition are of great importance. While in aerosol techniques particle size distributions of gas-borne particles are determined online, their elemental composition is commonly analyzed offline after an appropriate sampling and preparation procedure. To obtain both types of information online and simultaneously, a hyphenated setup was recently developed, including a Scanning Mobility Particle Sizer (SMPS) and an Inductively Coupled Plasma Mass Spectrometer (ICPMS). This allows first to classify the particles with respect to their mobility diameter, and then to determine their number concentration and elemental composition in parallel. A Rotating Disk Diluter (RDD) is used as the introduction system, giving more flexibility regarding the use of different aerosol sources. In this work, a practical guide is provided describing the different steps for establishing this instrumentation, and how to use this analysis tool. The versatility of this hyphenated technique is demonstrated in example measurements on three different aerosols generated out of a) a salt solution, b) a suspension, and c) emitted by a thermal process.

A Practical Guide on Coupling a Scanning Mobility Sizer and Inductively Coupled Plasma Mass Spectrometer SMPS-ICPMS

In this work a practical guide is provided, describing the different steps to establish the coupling of SMPS and ICPMS systems, and how to use them. Three descriptive examples are presented.

스캐닝 모빌리티 사이 저 및 유도 결합 플라스마 질량 분석기 (SMPS-ICPMS)의 결합에 관한 실용 가이드

A large variety of analytical methods are available to characterize particles in aerosols and suspensions. The choice of the appropriate technique depends ...

연구

화학

Scanning Light Scattering Profiler (SLPS) Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses

The scanning light scattering profiler (SLSP) methodology has been developed for the full-angle quantitative evaluation of forward and backward light scattering from intraocular lenses (IOLs) using goniophotometer principles. This protocol describes the SLSP platform and how it employs a 360&#176; rotational photodetector sensor that is scanned around an IOL sample while recording the intensity and location of scattered light as it passes through the IOL medium. The SLSP platform can be used to predict, non-clinically, the propensity for current and novel IOL designs and materials to induce light scatter. Non-clinical evaluation of light scattering properties of IOLs can significantly reduce the number of patient complaints related to unwanted glare, glistening, optical defects, poor image quality, and other phenomena associated with the unintended light scattering. Future studies should be conducted to correlate SLSP data with clinical results to help identify which measured light scatter is most problematic for patients that have undergone cataract surgery subsequent to IOL implantation.

Scanning Light Scattering Profiler SLPS Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses

This protocol describes the scanning light scattering profiler (SLSP) that enables the full-angle quantitative evaluation of forward and backward scattering of light from intraocular lenses (IOLs) using goniophotometer principles.

SLPS (Scanning Profiling Profiler) 기반 방법론으로 안구 내 렌즈의 전방 및 후방 산란을 정량적으로 평가

The scanning light scattering profiler (SLSP) methodology has been developed for the full-angle quantitative evaluation of forward and backward light ...

공학

Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living cells in real time. In this protocol, we cover the fundamental steps to realize an iSCAT microscope and complement it with additional imaging channels to monitor the viability of a cell under study. Following this, we use the method for real-time detection of single proteins as they are secreted from a living cell which we demonstrate with an immortalized B-cell line (Laz388). Necessary steps concerning the preparation of microscope and sample as well as the analysis of the recorded data are discussed. The video protocol demonstrates that iSCAT microscopy offers a straightforward method to study secretion at the single-molecule level.

We present a protocol for the real-time optical detection of single unlabeled proteins as they are secreted from living cells. This is based on interferometric scattering (iSCAT) microscopy, which can be applied to a variety of different biological systems and configurations.

레이블 무료 이미징의 단일 단백질 분 비 iSCAT 현미경 검사 법을 통해 살아있는 세포에서

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

골수 세포 역학의 실시간 영상<em&gt; APC<sup&gt; 최소 / +</sup</em&gt; 소장 종양 디스크 공 초점 현미경을 회전시켜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

면역학 및 감염병학

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

등방성 라이트 시트 현미경 검사 및 자동 세포 계보 분석을 통해 아닌 간 분해능을 통한 배아 발생 발생

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

발생학

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

구조화 조명 현미경 데이터의 시공간적 이미지 상관 분광학에 의해 정량화 T 세포에서 대뇌 피질 굴지 흐름

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

실시 한 형광 현미경으로 여러 영상 모드

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

생체공학

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and &#62; 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

입체 슈퍼의 간섭 광 활성화 현지화 현미경으로 F-액틴 필라멘트의 해상도 현미경 (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

생물학

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

Confocal 현미경 검사 법 밝혀 세포 표면 수용 체 이미지 상관 분광학을 통해 집계

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

Live-cell Imaging of Single-Cell Arrays (LISCA) - a Versatile Technique to Quantify Cellular Kinetics

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high throughput. In general, the acquisition of single-cell time courses from cultured cells is hampered by cell motility and diversity of cell shapes. Adhesive micro-arrays standardize single-cell conditions and facilitate image analysis. LISCA combines single-cell microarrays with scanning time-lapse microscopy and automated image processing. Here, we describe the experimental steps of taking single-cell fluorescence time courses in a LISCA format. We transfect cells adherent to a micropatterned array using mRNA encoding for enhanced green fluorescent protein (eGFP) and monitor the eGFP expression kinetics of hundreds of cells in parallel via scanning time-lapse microscopy. The image data stacks are automatically processed by newly developed software that integrates fluorescence intensity over selected cell contours to generate single-cell fluorescence time courses. We demonstrate that eGFP expression time courses after mRNA transfection are well described by a simple kinetic translation model that reveals expression and degradation rates of mRNA. Further applications of LISCA for event time correlations of multiple markers in the context of signaling apoptosis are discussed.

Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics

We present a method for the acquisition of fluorescence reporter time courses from single cells using micropatterned arrays. The protocol describes the preparation of single-cell arrays, the setup and operation of live-cell scanning time-lapse microscopy and an open-source image analysis tool for automated preselection, visual control and tracking of cell-integrated fluorescence time courses per adhesion site.

세포 역학을 정량화하는 다목적 기술인 단세포 어레이(LISCA)의 라이브 셀 이미징

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high ...