전립선 세포주 친절 암 센터, UC 데이비스 박사 링 - 유 왕 박사 싱 - 지엔 쿵에 의해 제공됩니다. 이 프로젝트는 NIH의 galvanotaxis 부여 4R33AI080604에 의해 지원됩니다.

1. 배양 전립선 세포 <ol><li> 문화 PRNS-1-1 5 % CO 2, 37 ℃에서 10 % FBS와 항생제 - 안티 곰팡이로 보충 된 RPMI 1640 배지에서 100mm 배양 접시에 PNT2 및 전립선 세포. 세포 galvanotaxis 실험에 80 % 포화 상태에 도달 할 때까지 매일 배양 배지를 새로. </li></ol> 2. 조립 Galvanotaxis 챔버 <ol><li> 하단 챔버 조립 <ol><li> 2- 프로판올와 플라스틱 galvanotaxis 챔버를 닦습니다. 주사기와 챔버의 하부 측에 원형의 개구 주변 해양 급 실리콘 실러를 적용하고 큰 커브 글라스 (도 1)에 부착. 커버 글라스를 아래로 밀어 면봉으로 초과 접착제를 닦아면 주걱의 뒷면을 사용합니다. 원형 개구부는 중간 전류는 두 매질 내부 저장소 사이에 위치 된 세포 흐르게. </li><li> 6mm × 2를 만들기 위해 작은 커버 글라스를 잘라다이아몬드 포인트 마커 5mm 스페이서. </li><li> 챔버를 뒤집습니다. 주사기 및 / 또는 하단 유리에 2 원형 구멍 사이의 세포 부착을위한 10mm X 25mm 채널 표면을 만들 수있는 금속 주걱 실리콘 접착제의 2 줄무늬를 적용합니다. 이 원형 구멍 사이에 유리 스페이서 2 개를 붙인다. 스페이서를 아래로 밀어 면봉 및 / 또는 이쑤시개와 초과 접착제를 닦아면 어플리케이터의 뒷면을 사용합니다. </li><li> 실리콘 접착제가 경화 될 때까지 24 시간 동안 챔버를 건조. 접착제에서 아세트산 잔기를 제거 증류수에 밤새 챔버를 담근다. 즉시 사용 가능한 챔버를 건조, 또는 깨끗한 용기에 나중에 사용하기 위해 챔버를 저장합니다. <img alt="그림 1" fo:content-width="5in" src="/files/ftp_upload/51973/51973fig1highres.jpg" width="500" /> 그림 1 : 하단 galvanotaxis 챔버의 조립. A)가 큰 커브 글라스) T 세정 챔버. B의 하단에 부착된다유리 스페이서의 조각이 원형 구멍 사이에 접착되어 하시다 세포 부착을위한 10mm X 25mm 채널을 만들 수 있습니다. </li></ol></li><li> galvanotaxis 챔버에 세포를 심는 <ol><li> 실험에 필요한 galvanotaxis 챔버의 필요 수를 준비합니다. 각 세포주 또는 치료는 하나의 galvanotaxis 챔버가 필요합니다. 2- 프로판올과 galvanotaxis 챔버를 닦습니다. 멸균 PBS의 3 배에 실을 세척하고 챔버에서 2 원형 구멍 사이의 액체 흐름을 확인합니다. </li><li> 무균 세포 배양 접시에있는 챔버를 동봉하고 15 분 동안 37 ℃에서 평형 수 있습니다. 5 분 동안 37 ° C에서 5 ml의 0.25 % 트립신 EDTA를 사용하여 배양 접시에서 전립선 세포를 분리. PBS에 5 ㎖의 10 % FBS와 트립신 EDTA를 중화. </li><li> 15 ㎖의 튜브에 세포를 옮기고 200 XG, 37 ° C에서 5 분 동안 원심 분리하여 세포. 상등액을 대기음 배지 1 ㎖에 세포를 다시 일시. </li><li> 20 μ를 타고셀 L의 용액은 카운팅 챔버에로드하고 세포 수를 산출한다. 문화 매체와 8 × 104 세포 / ㎖로 세포 농도를 조정합니다. </li><li> 37 ° C 배양기에서 galvanotaxis 챔버를 가져 가라. 각 챔버에 세포 현탁액 350 μl를 시드. </li><li> 킴 와이프 또는 습윤 5 % CO 2, 37 ℃에서 습도 인큐베이터 챔버에서 배양 접시에서 밤새 챔버에서 세포를 배양한다. </li></ol></li></ol><img alt="그림 2" fo:content-width="5in" src="/files/ftp_upload/51973/51973fig2highres.jpg" width="500" /> 그림 2. 챔버에 세포를 시드 하부 측실을 건조 세정 A) 전립선 세포를 트립신 카운트 챔버로 이송하고, 밤새 인큐베이션 B) 상단 커버 글라스는 촬영 전에 챔버를 밀봉하도록 부착된다... <ol start="3"><li>상단 챔버 ssembling <ol><li> 촬영하기 전에 상단 챔버를 조립합니다. 현미경은 한 번에 하나의 챔버 galvanotaxis 촬상 수용 할 수있다. 각 galvanotaxis 챔버, 37 ° C에서 배양 배지 10 ㎖ 워밍업. 37 ° C 온난화 판에 인큐베이터에서 galvanotaxis 챔버를 전송합니다. </li><li> 부착되지 않은 세포를 제거하기 위해 문화 매체와 세포를 씻어. 챔버 내에서 매체의 400 μl를 남겨주세요. 모두 유리 스페이서의 상단에 높은 진공 그리스를 적용 할 주사기를 사용합니다. </li><li> 챔버를 밀봉하는 작은 커버 글라스를 추가합니다. 부드럽게면 주걱의 뒷면에 커버 글라스를 누르십시오. 면봉과 초과 매체를 닦아냅니다. </li><li> 유리 표면을 건조하고, 커버 글라스와 챔버 사이의 간격을 밀봉 높은 진공 그리스를 적용합니다. 그리스를 확산 금속 주걱을 사용합니다. 내부 저수지에 배지 4 ML을 추가하고 저수지 사이의 액체 흐름을 확인합니다. </li></ol></li><li> 한천 만들기다리 <ol><li> 2 인치 길이의 PVC 튜브의 한 쌍을 잘라 (16분의 503 (내경) x 5/16 (외경) x 1/16 월), 플립과 100 비이커에 삽입합니다. </li><li> 200mg의 박토 - 한천을 측정하고 2 % 한천 겔을 10 ml의 배지와 함께 50 ml의 플라스크에 추가한다. 30 초 동안 전자 레인지. 전송 피펫과 플라스틱 튜브에 한천 겔을로드합니다. 고형화 한천을 10 분 동안 실온에서 한천 교량 남기기. </li><li> galvanotaxis 챔버의 외부 저수지에 2 ml의 배지를 추가합니다. 전류를 전도하기 위해 내부 및 외부 매체 저수지에 한천 다리를 삽입합니다. </li></ol></li></ol><img alt="그림 3" fo:content-width="3.5in" src="/files/ftp_upload/51973/51973fig3highres.jpg" width="350" /> 그림 3 :. 챔버에 세포를 얇은 껍질이 그림은 galvanotaxis 챔버의 최종 조립을 보여합니다. 챔버는 완전히 고 진공 그리스로 밀봉된다. 중간 t을 추가오 저수지를 작성하고 두 한천 교량 챔버에 삽입됩니다. 이어서 galvanotaxis 챔버는 현미경 스테이지에 전달되고, 전극은 전기장을 적용하도록 부착된다. 조립 galvanotaxis 챔버의 측면도는 전류가 한천 교량과 커버 유리 사이의 공간을 통해 흐르고 셀 위에 함을 입증하기에 도시된다. 3. 시간 경과 영상 <ol><li> 이전 영상에 공기 순환 2 시간에서 5 % CO 2와 37 ° C에 환경 챔버에 전환합니다. </li><li> 현미경에 전환하고 이미지 획득 소프트웨어를 시작합니다. 현미경 스테이지를 보정하고, 10 배 목표를 선택합니다. </li><li> 현미경 무대에 galvanotaxis 챔버를 전송 테이프로 실을 확보하고 세포에 초점을 맞 춥니 다. 오른쪽에 음극과 외부 저수지에 galvanotaxis 전극을 삽입합니다. 와이어와 테이프로 전극을 고정합니다. </li><li> 전력 보에 스위치X는 챔버에 전계를 적용한다. 25mm 긴 챔버 (100 MV / mm) 2.5 볼트에 도달 할 수 챔버에서 전압계와 출력 전압을 측정합니다. 출력 전류를 조절하여 실험 기간 동안 전계 강도를 유지한다. </li><li> 열 시간 경과 영화를 생성하는 소프트웨어의 필름과 챔버에서 10 점을 선택합니다. 2 시간 동안 10 분 간격으로 인수 조건을 설정합니다. </li><li> 촬영을 시작하고, 필요에 따라 출력 전류를 조정한다. </li><li> 실험의 마지막 단계에서 챔버를 제거하고 95 % 알코올로 세포를 고정한다. 청소 면도날 열린 챔버 휴식하고 다시 사용하기. </li></ol> Galvanotaxis 4. 정량화 <ol><li> 시간 경과 영화와 다시의 방향 이미지의 상단에 음극을 돌립니다. 셀 추적 소프트웨어에 영화를 내 보냅니다. </li><li> 수동으로 각 동영상 (도 4 A, B)에서 각각의 시간 지점에서 10-20 세포 (x, y)의 위치를 추적.남북 방향에 대하여 이주 거리 및 각도는, 전기장의 방향은 동일한, 셀 추적 소프트웨어 (도 4c)에서 산출한다. 주 : 평균 이동 속도가 총 이동 거리와 총 촬영 시간으로 변환됩니다. 방향성은 코사인 값 각도에서 변환된다. 세포가 임의의 방향성으로 마이그레이션하는 경우, 순 코사인의 평균은 제로에 가까운 것입니다. 세포가 음극쪽으로 이동하는 경우 직접, 코사인 값은 1이 될 것이다. 세포를 직접 애노드 측으로 이동하는 경우, 코사인 값은 -1이 될 것이다. </li></ol><img alt="그림 4" fo:content-width="4in" src="/files/ftp_upload/51973/51973fig4highres.jpg" width="400" /> 도 4 : 셀 추적 방향성을 측정하기 위해 세포 추적 이미지 라인) 오버레이.. 셀 (X, Y) 위치는 manua 아르에서야 시간 경과 영화에서 추적. . 세포는 임의로 이동하면 평균 코사인 제로에 가까운 B) 그러나, 세포가 음극 또는 양극을 향해 이동하는 경우, 평균 코사인 값 + (음극) 또는 부근 -. (양극) 1.0 C ) 방향성은 마이그레이션 각도 (θ)에서 변환 된 코사인 값에 의해 제공됩니다. 코사인 (θ)은 거리 B (전계의 방향에 투영 거리)까지의 거리 (이동 거리)의 비율과 같다. <ol start="3"><li> 측정 값을 저장합니다. 조합 된 결과를 계산하는 데이터베이스 어플리케이션에 데이터를 가져. </li><li> 막대 그래프 (그림 5) 음모 스프레드 시트에 평균 이동 속도와 평균 코사인 값의 결합 된 데이터를 보냅니다. </li></ol>

<ol>
	<li>Reid, B., Nuccitelli, R., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+measurement+of+bioelectric+currents+with+a+vibrating+probe.">Non-invasive measurement of bioelectric currents with a vibrating probe.</a> Nat Protoc. 2 (3), 661-669 (2007).</li><li>Cao, L., 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=Endogenous+electric+currents+might+guide+rostral+migration+of+neuroblasts.">Endogenous electric currents might guide rostral migration of neuroblasts.</a> EMBO Rep. 14 (2), 184-190 (2013).</li><li>Hotary, K. B., Robinson, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+electrical+currents+and+voltage+gradients+in+Xenopus+embryos+and+the+consequences+of+their+disruption.">Endogenous electrical currents and voltage gradients in Xenopus embryos and the consequences of their disruption.</a> Dev Biol. 166 (2), 789-800 (1994).</li><li>Hotary, K. B., Robinson, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+a+role+for+endogenous+electrical+fields+in+chick+embryo+development.">Evidence of a role for endogenous electrical fields in chick embryo development.</a> Development. 114 (4), 985-996 (1992).</li><li>Yamashita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+axon+guidance+in+embryonic+retina:+galvanotropism+revisited.">Electric axon guidance in embryonic retina: galvanotropism revisited.</a> Biochem Biophys Res Commun. 431 (2), 280-283 (2013).</li><li>Wood, M. D., Willits, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applied+electric+field+enhances+DRG+neurite+growth:+influence+of+stimulation+media,+surface+coating+and+growth+supplements.">Applied electric field enhances DRG neurite growth: influence of stimulation media, surface coating and growth supplements.</a> J Neural Eng. 6 (4), 046003 (2009).</li><li>Kucerova, 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=The+role+of+electrical+signals+in+murine+corneal+wound+re-epithelialization.">The role of electrical signals in murine corneal wound re-epithelialization.</a> J Cell Physiol. 226 (6), 1544-1553 (2011).</li><li>Sillman, A. L., Quang, D. M., Farboud, B., Fang, K. S., Nuccitelli, R., Isseroff, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Dermal+fibroblasts+do+not+exhibit+directional+migration+on+collagen+I+in+direct-current+electric+fields+of+physiological+strength.">Human Dermal fibroblasts do not exhibit directional migration on collagen I in direct-current electric fields of physiological strength.</a> Exp Dermatol. 12 (4), 396-402 (2003).</li><li>Allen, G. M., Mogilner, A., Theriot, 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=Electrophoresis+of+cellular+membrane+components+creates+the+directional+cue+guiding+keratocyte+galvanotaxis.">Electrophoresis of cellular membrane components creates the directional cue guiding keratocyte galvanotaxis.</a> Curr Biol. 23 (7), 560-568 (2013).</li><li>Nishimura, K. Y., Isseroff, R. R., Nuccitelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+keratinocytes+migrate+to+the+negative+pole+in+direct+current+electric+fields+comparable+to+those+measured+in+mammalian+wounds.">Human keratinocytes migrate to the negative pole in direct current electric fields comparable to those measured in mammalian wounds.</a> J Cell Sci. 109 (1), 199-207 (1996).</li><li>Farboud, B., Nuccitelli, R., Schwab, I. R., Isseroff, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DC+electric+fields+induce+rapid+directional+migration+in+cultured+human+corneal+epithelial+cells.">DC electric fields induce rapid directional migration in cultured human corneal epithelial cells.</a> Exp Eye Res. 70 (5), 667-673 (2000).</li><li>Fang, K. S., Ionides, E., Oster, G., Nuccitelli, R., Isseroff, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+receptor+relocalization+and+kinase+activity+are+necessary+for+directional+migration+of+keratinocytes+in+DC+electric+fields.">Epidermal growth factor receptor relocalization and kinase activity are necessary for directional migration of keratinocytes in DC electric fields.</a> J Cell Sci. 112 (12), 1967-1978 (1999).</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=Activated+T+lymphocytes+migrate+toward+the+cathode+of+DC+electric+fields+in+microfluidic+devices.">Activated T lymphocytes migrate toward the cathode of DC electric fields in microfluidic devices.</a> Lab Chip. 11 (7), 1298-1304 (2011).</li><li>Meng, X., Arocena, M., Penninger, J., Gage, F. H., Zhao, M., Song, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+mediated+electrotaxis+of+embryonic+and+adult+neural+progenitor+cells+in+the+presence+of+growth+factors.">PI3K mediated electrotaxis of embryonic and adult neural progenitor cells in the presence of growth factors.</a> Exp Neurol. 227 (1), 210-217 (2011).</li><li>Wu, D., Ma, X., Lin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DC+Electric+Fields+Direct+Breast+Cancer+Cell+Migration,+Induce+EGFR+Polarization,+and+Increase+the+Intracellular+Level+of+Calcium+Ions.+.">DC Electric Fields Direct Breast Cancer Cell Migration, Induce EGFR Polarization, and Increase the Intracellular Level of Calcium Ions. .</a> Cell Biochem Biophys. 67 (3), 1115-1125 (2013).</li><li>Martin-Granados, 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=A+role+for+PP1/NIPP1+in+steering+migration+of+human+cancer+cells.">A role for PP1/NIPP1 in steering migration of human cancer cells.</a> PLoS One. 7 (7), 40769 (2012).</li><li>Yang, H. Y., Charles, R. P., Hummler, E., Baines, D. L., Isseroff, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epithelial+sodium+channel+mediates+the+directionality+of+galvanotaxis+in+human+keratinocytes.">The epithelial sodium channel mediates the directionality of galvanotaxis in human keratinocytes.</a> J Cell Sci. 126 (9), 1942-1951 (2013).</li><li>Zhao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+signals+control+wound+healing+through+phosphatidylinositol-3-OH+kinase-gamma+and+PTEN.">Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-gamma and PTEN.</a> Nature. 442 (7101), 457-460 (2006).</li><li>Shanley, L. J., Walczysko, P., Bain, M., MacEwan, D. J., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influx+of+extracellular+Ca2++is+necessary+for+electrotaxis+in+Dictyostelium.">Influx of extracellular Ca2+ is necessary for electrotaxis in Dictyostelium.</a> J Cell Sci. 119 (22), 4741-4748 (2006).</li><li>Zhao, M., Agius-Fernandez, A., Forrester, J. V., McCaig, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+migration+of+corneal+epithelial+sheets+in+physiological+electric+fields.">Directed migration of corneal epithelial sheets in physiological electric fields.</a> Invest Ophthalmol Vis Sci. 37 (13), 2548-2558 (1996).</li><li>Peng, H. B., Jaffe, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization+of+fucoid+eggs+by+steady+electrical+fields.">Polarization of fucoid eggs by steady electrical fields.</a> Dev Biol. 53 (2), 277-284 (1976).</li><li>Lee, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+adult+human+prostatic+epithelial-cells+immortalized+by+polybrene-induced+DNA+transfection+with+a+plasmid+containing+an+origin-defective+sv40-genome.">Characterization of adult human prostatic epithelial-cells immortalized by polybrene-induced DNA transfection with a plasmid containing an origin-defective sv40-genome.</a> Int J Oncol. 4 (4), 821-830 (1994).</li><li>Berthon, P., Cussenot, O., Hopwood, L., Leduc, A., Maitland, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+expression+of+sv40+in+normal+human+prostatic+epithelial+and+fibroblastic+cells+-+differentiation+pattern+of+non-tumorigenic+cell-lines.">Functional expression of sv40 in normal human prostatic epithelial and fibroblastic cells - differentiation pattern of non-tumorigenic cell-lines.</a> Int J Oncol. 6 (2), 333-343 (1995).</li><li>Aurich-Costa, J., Vannier, A., Gr&#233;goire, E., Nowak, F., Cherif, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IPM-FISH,+a+new+M-FISH+approach+using+IRS-PCR+painting+probes:+application+to+the+analysis+of+seven+human+prostate+cell+lines.">IPM-FISH, a new M-FISH approach using IRS-PCR painting probes: application to the analysis of seven human prostate cell lines.</a> Genes Chromosomes Cancer. 30 (2), 143-160 (2001).</li><li>Tyson, D. R., Inokuchi, J., Tsunoda, T., Lau, A., Ornstein, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+requirements+of+prostatic+epithelial+cell+lines+for+acinar+morphogenesis+and+lumen+formation+in+vitro:+role+of+extracellular+calcium.">Culture requirements of prostatic epithelial cell lines for acinar morphogenesis and lumen formation in vitro: role of extracellular calcium.</a> Prostate. 67 (15), 1601-1613 (2007).</li><li>Lang, S. H., Sharrard, R. M., Stark, M., Villette, J. M., Maitland, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostate+epithelial+cell+lines+form+spheroids+with+evidence+of+glandular+differentiation+in+three-dimensional+Matrigel+cultures.">Prostate epithelial cell lines form spheroids with evidence of glandular differentiation in three-dimensional Matrigel cultures.</a> Br J Cancer. 85 (4), 590-599 (2001).</li><li>Babona-Pilipos, R., Popovic, M. R., Morshead, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+galvanotaxis+assay+for+analysis+of+neural+precursor+cell+migration+kinetics+in+an+externally+applied+direct+current+electric+field.">A galvanotaxis assay for analysis of neural precursor cell migration kinetics in an externally applied direct current electric field.</a> J Vis Exp. (68), (2012).</li><li>Meng, X., 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=Electric+field-controlled+directed+migration+of+neural+progenitor+cells+in+2D+and+3D+environments.">Electric field-controlled directed migration of neural progenitor cells in 2D and 3D environments.</a> J Vis Exp. (60), (2012).</li><li>Pullar, C. E., Isseroff, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+mediates+keratinocyte+directional+migration+in+an+electric+field.">Cyclic AMP mediates keratinocyte directional migration in an electric field.</a> J Cell Sci. 118 (9), 2023-2034 (2005).</li><li>Sheridan, D. M., Isseroff, R. R., Nuccitelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imposition+of+a+physiologic+DC+electric+field+alters+the+migratory+response+of+human+keratinocytes+on+extracellular+matrix+molecules.">Imposition of a physiologic DC electric field alters the migratory response of human keratinocytes on extracellular matrix molecules.</a> J Invest Dermatol. 106 (4), 642-646 (1996).</li><li>Feng, J. 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=Guided+migration+of+neural+stem+cells+derived+from+human+embryonic+stem+cells+by+an+electric+field.">Guided migration of neural stem cells derived from human embryonic stem cells by an electric field.</a> Stem Cells. 30 (2), 349-355 (2012).</li><li>Mukerjee, E. V., Isseroff, R. R., Nuccitelli, R., Collins, S. D., Smith, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microneedle+array+for+measuring+wound+generated+electric+fields.">Microneedle array for measuring wound generated electric fields.</a> Conf Proc IEEE Eng Med Biol Soc. 1, 4326-4328 (2006).</li><li>Nuccitelli, R., Nuccitelli, P., Li, C., Narsing, S., Pariser, D. M., Lui, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electric+field+near+human+skin+wounds+declines+with+age+and+provides+a+noninvasive+indicator+of+wound+healing.">The electric field near human skin wounds declines with age and provides a noninvasive indicator of wound healing.</a> Wound Rep. and Reg. 19, 645-655 (2011).</li></ol>

The authors have nothing to disclose.

셀의 galvanotaxis 응답의 분석은 많은 셀룰러 이동성 또는 성장을위한 중요한 기능 표시기 (27), (28)를 처리했다. 여기서 우리는 두 전립선 세포주 영화를 유리 표면에 특별 주문품 실을 사용한다. 이들 세포주 galvanotaxis 상이한 정도를 보여, 우리는 세포 내 위치 파악 또는 galvanotaxis - 매개 단백질의 활성화는 galvanotaxis 응답의 관측 차이의 결과로, 불멸 세포주를 생성하는 과정을 방해 할 수 있...

내인성 전기장 피부 1, 32, 33, 2와 같은 다양한 뇌 조직에서 검출된다. 생리 전계 신경 공정 5, 6의 생장을 유도하고 상피 각막 상처 봉합 1,7 촉진 지향, 배 발달 3,4- 포함한 특정 생물학적 기능을 제공. 시험관 내 배양 된 세포에 직류 전계인가 모방 생리 전기장을하고 방향 세포의 이동, 또는 galvanotaxis를 유도한다. Galvanotaxis는 neuroblasts 2, 신경 전구 세포 (14), 섬유 아세포 8, 물고기 각질 9, 인간 상피와 각막 각질 10-12 연구 13 림프구되었습니다. (-) 극인가 필드에 노출되는 경우, 세포 연구의 대부분은 방향성 cathodal 향해 이동한다. 그러나, 고도 등 여러 전이성 암세포인간의 유방암 세포와 인간 전립선 암 세포주 PC-3M이 anodal (+) 극 (15, 16)로 이동한다. 여러 메커니즘 galvanotaxis을 중재 또는 활성화를 포함하는 전기장을 감지하는 세포의 능력을 설명하기 위해 제안 EGF 12 수용체, 상피 나트륨 채널 (17)는 PI3K 및 PTEN (18), 및 칼슘의 방출이기구는 아직 완전히 이해되지. 15 (19) 이온 및 그것은 여러 신호 전달 경로가 galvanotaxis에 참여하는 것이 가능하다. 여기 보여 2 차원 galvanotaxis 방법은 점착성, 운동성 세포의 방향 이동을 특성화하기 위해, 하나 (18), (20).이 기법에서 수정 된 융합 세포의 시트를 개개의 세포 이동을 10, 12, 17 또는 이동을 모니터링하는 데 유용 펭과 제피 (21), 그리고 니시무라 등. 주문 제작, 명확한 PVC 챔버 10, 이동식 coversl와IPS와 같은 면역 형광 이미징 차 분석을위한 galvanotaxis, 후 쉽게 셀 검색을 허용. galvanotaxis 챔버의 유리 표면은 고배율과 형광 표지 된 세포와 촬영을 허용하는 광학 호환이다. 그것은 또한 표면 코팅 또는 전하 변경과 같은, 유리의 표면 개질과 실험 설계를 허용한다. 제 1 커버 글라스로 이루어지는 스페이서 위에 셀 전류 흐름을 최소화하기 위해 챔버에 사용된다; 따라서, 전류 흐름의 제곱에 비례 주울 가열, 실험 기간 동안 세포를 과열 않을 것이다. 한천 연결 교량 셀 전극의 직접 접촉을 방지하고 galvanotaxis 동안 매체의 pH 나 이온 농도의 변화를 방지한다. 두 개의 비 - 인간 종 양성 전립선 세포주는이 연구에서 galvanotaxis 그들의 응답을 조사 하였다. PRNS-1-1 22 PNT2 (23)는 모두 SV4 있습니다성장 인자 의존적 세포주는 전립선 특이 항원 (PSA)의 낮은 또는 전혀 식 5, 8, 18, 19 아세포 상피 마커를 발현하는, 0 - 불멸화. 두 세포주 정상 상피 세포의 다각형 형태를 유지하지만, 염색체 이상은 핵형 (22, 24)에서 관찰되었다. PRNS-1-1과 PNT2 대부분의 실험에서 유사한 행동을 공유하지만, 그들이 선포 구조의 형성과의 차이를 표시 할 galvanotaxis. 3-D 행렬에, 마트 리겔은 PRNS-1-1 세포는 정상적인 전립선 조직 (25)을 닮은 루멘 선포의 중공 구조를 형성한다. 그러나 PNT2 세포 내강 상피 (26) 또는 편광없이 고체 스페 로이드를 형성한다. PRNS-1-1 세포는 또한 현재 연구 PNT2보다 높은 galvanotactic 응답을 보여준다. 선포의 구조의 형성 및 galvanotaxis 간의 상관 관계는 PRNS-1-1 galvanotactic 신호 (PR)를 조직에서 역할을 할 수 있음을 시사ostate 선 조직 내생 전기장에 대응 움직임, 그리고이 두 세포주를 구별하기 위해 추가 특성을 제공한다.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Cells&lt;/strong&gt;</td></tr><tr><td>pRNS-1-1 prostate cells</td><td></td><td></td><td>Lee, &lt;em&gt;et al.&lt;/em&gt; (1994)</td></tr><tr><td>PNT2 prostate cells</td><td>Sigma-Aldrich</td><td>95012613-1VL</td><td>Berthon, &lt;em&gt;et al.&lt;/em&gt; (1995)</td></tr><tr><td colspan="4">&lt;strong&gt;Medium and solutions&lt;/strong&gt;</td></tr><tr><td>RPMI 1640 medium&amp;nbsp;</td><td>Invitrogen</td><td>11875-093</td><td>warm up to 37 &amp;deg;C before use</td></tr><tr><td>Fetal Bovine Serum - Premium</td><td>Atlanta Biologicals</td><td>S11150</td><td>10% in PBS, warm up to 37 &amp;deg;C before use</td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>Life Technologies</td><td>15240</td><td>add 5 ml to 500 ml medium</td></tr><tr><td>2-propanol</td><td>VWR</td><td>BDH1133-5GL</td><td></td></tr><tr><td>PBS</td><td>-</td><td>-</td><td>137 mM NaCl, 2.7 mM KCl, 4.3 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; and 1.5 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; in 1,000 ml of H&lt;sub&gt;2&lt;/sub&gt;O, pH to 7.4 and autoclaved, warm up to 37 &amp;deg;C before use</td></tr><tr><td>0.25% Trypsin-EDTA&amp;nbsp;</td><td>Invitrogen</td><td>25200-056</td><td>warm up to 37 &amp;deg;C before use, treat cells for 3-5 min at 37 &amp;deg;C</td></tr><tr><td colspan="4">&lt;strong&gt;Galvanotaxis device&lt;/strong&gt;</td></tr><tr><td>Galvanotaxis chambers</td><td>Precision Plastics Inc, CA</td><td></td><td>Custom-designed (1/4&quot; x 2&quot; x 3.5&quot;), non-toxic, clear PVC chambers. Please contact the authors for the design specs.</td></tr><tr><td>Galvanotaxis electrodes</td><td>UCD electric shop</td><td></td><td>platinum coiled electrodes with flexable cords</td></tr><tr><td>Galvanotaxis power box</td><td>Substrate Engineering, CA</td><td></td><td>custom-designed DC power output with voltmeter</td></tr><tr><td>Microscope Cover Glass, Large, 45 x 50 mm, No. 1.5</td><td>Fisher</td><td>12-544-F</td><td></td></tr><tr><td>Microscope Cover Glass, small, 25 x 25 mm, No. 1</td><td>ThermoScientific</td><td>3307</td><td></td></tr><tr><td>Diamond point marker</td><td>ThermoScientific</td><td>750</td><td></td></tr><tr><td>Marine grade silicon sealer, clear</td><td>3M</td><td>051135-08019</td><td></td></tr><tr><td>High vacuum grease</td><td>Dow Corning</td><td>2021846-0807</td><td></td></tr><tr><td>6 ml syringe</td><td>Fisher Scientific</td><td>05-561-64</td><td></td></tr><tr><td>Nichiryo Syringe, 1.5 ml</td><td>Nichiryo</td><td>SG-M</td><td></td></tr><tr><td>Cotton applicators</td><td>Purtian Medical Products</td><td>806-WC</td><td></td></tr><tr><td>Qtips</td><td>Johnson &amp;amp; Johnson</td><td>729389</td><td></td></tr><tr><td>Nalgene 180 PVC tubing&amp;nbsp;</td><td>Nalgene</td><td>8000-9030</td><td>503/16 ID x 5/16 OD x 1/16 Wall</td></tr><tr><td>Bacto-Agar</td><td>Difco</td><td>0140-01</td><td>make 2% agar solution</td></tr><tr><td>Razor Blade</td><td>Personna</td><td>74-0001</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipments and Software&lt;/strong&gt;</td></tr><tr><td>Benchtop Centrifuge</td><td>Eppendorf</td><td>5810R</td><td>operated with an A-4-62 rotor</td></tr><tr><td>Cellometer Auto T4</td><td>Nexcelom</td><td>Auto T4</td><td></td></tr><tr><td>Cellometer counting chambers</td><td>Nexcelom</td><td>CHT4-SD100-002</td><td>load 20 &amp;#956;l cell solutions to count</td></tr><tr><td>Culture Temp Warming plate</td><td>Bel-Art Scienceware</td><td>370150000</td><td>to keep the galvanotaxis chambers at 37 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Eclipse TE-2000 microscope with motorized stage and environmental chamber</td><td>Nikon</td><td></td><td></td></tr><tr><td>Plan Fluor 10X/0.30 objective len</td><td>Nikon</td><td></td><td></td></tr><tr><td>Retiga EX CCD camera&amp;nbsp;</td><td>Qimaging</td><td></td><td>Cooled CCD camara, mono-color, 12-bit</td></tr><tr><td>Compressed air with 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Airgas</td><td></td><td>special order</td></tr><tr><td>Volocity 6.3</td><td>PerkinElmer</td><td></td><td>Image acquiring software</td></tr><tr><td>Improvision OpenLab 5.5.2</td><td>PerkinElmer</td><td></td><td>Cell tracking software and customized to measure migration angles</td></tr><tr><td>FileMaker Pro Advanced, 8.0</td><td>FileMaker</td><td></td><td></td></tr><tr><td>Microsoft Excel 2008 for Mac</td><td>Microsoft</td><td></td><td></td></tr></tbody></table>

utilizing custom-designed galvanotaxis chambers to study directional migration of prostate cells

생리 전기장은 배아 발달, 신경 가지 상피 상처 치유에 세포의 이동을 지시하는 등 특정 생물학적 기능을 제공합니다. 시험 관내에서 배양 된 세포에 직류 전계를인가하여 세포의 이동 방향, 또는 galvanotaxis을 유도한다. 여기 보여 2 차원 galvanotaxis 방법은 주문 제작 된 폴리 (비닐 클로라이드) (PVC) 챔버, 유리 표면에, 백금 전극과 셀 묘화되는 동력 스테이지를 이용하여 수정된다. PVC 챔버 및 백금 전극은 낮은 세포 독성을 전시하고 저렴하고 다시 사용 가능한입니다. 유리 표면과 전동 현미경 스테이지는 이미지의 품질을 개선하고 세포에 유리 표면과 치료 가능한 수정을 허용한다. 우리는 두 가지 비 - 종양 형성, SV40-전립선 무한 증식 세포주, PRNS-1-1의 PNT2 galvanotaxis을 촬영. 이들 두 세포주는 유사한 이동 속도를 보여 모두를 향해 이동음극,하지만 그들은 galvanotaxis의 방향성의 다른 정도를 보여 않습니다. 이 프로토콜을 통해 얻어진 결과 PRNS-1-1 PNT2 세포주들은 지향성 응답 이동성을 관리 다른 고유 특성을 가질 수 있음을 시사한다.

전립선 세포 (PRNS-1-1과 PNT2)의 두 라인은이 방법으로 조사 하였다. 두 줄의 셀은 2 시간 (도 5a)에 걸쳐 1.0 +/- 0.3 미크론 / 분의 속도로 이동 유사한. 그러나, 전계 지향성 PRNS-1-1 선 0.7 +/- 0.3 및 PNT2 라인 (도 5B) 0.2 +/- 0.8이다. 결과들은 상이한 위치 단서 지향성 응답이 발생할 다른 세포 시그널링 메커니즘을 가지고 있음을 시사 (100 세포 추적 하였다 p &lt;0.01)이 두 세포주 galvano...

Watch this Scientific Journal Video about Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells at JoVE.com

Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells

We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.

전립선 세포의 방향 마이그레이션 연구에 맞춤 설계 Galvanotaxis 챔버를 활용

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and ...

utilizing-custom-designed-galvanotaxis-chambers-to-study-directional

Research

JoVE Journal

Biology

9.1K 조회수.  University of California, Davis. We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.

비디오: Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement membrane (BM) during age-related pathologies and metabolic disorders (e.g. cancer, diabetes, microvascular disease, retinopathy, nephropathy and neuropathy). The premise of the model is non-enzymatic crosslinking of reconstituted BM (rBM) matrix by treatment with glycolaldehyde (GLA) to promote advanced glycation endproduct (AGE) generation via the Maillard reaction. Examples of laboratory techniques that can be used to confirm AGE generation, non-enzymatic crosslinking and increased stiffness in GLA treated rBM are outlined. These include preparation of native rBM (treated with phosphate-buffered saline, PBS) and stiff rBM (treated with GLA) for determination of: its AGE content by photometric analysis and immunofluorescent microscopy, its non-enzymatic crosslinking by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) as well as confocal microscopy, and its increased stiffness using rheometry. The procedure described here can be used to increase the rigidity (elastic moduli, E) of rBM up to 3.2-fold, consistent with measurements made in healthy versus diseased human prostate tissue. To recreate the biophysical microenvironment associated with the aging and diseased prostate gland three prostate cell types were introduced on to native rBM and stiff rBM: RWPE-1, prostate epithelial cells (PECs) derived from a normal prostate gland; BPH-1, PECs derived from a prostate gland affected by benign prostatic hyperplasia (BPH); and PC3, metastatic cells derived from a secondary bone tumor originating from prostate cancer. Multiple parameters can be measured, including the size, shape and invasive characteristics of the 3D glandular acini formed by RWPE-1 and BPH-1 on native versus stiff rBM, and average cell length, migratory velocity and persistence of cell movement of 3D spheroids formed by PC3 cells under the same conditions. Cell signaling pathways and the subcellular localization of proteins can also be assessed.

Here we explain a protocol for modelling the biophysical microenvironment where crosslinking and increased stiffness of the basement membrane (BM) induced by advanced glycation endproducts (AGEs) has pathological relevance.

전립선 암과 기타 노화 관련 병리 또는 대사 질환의 생물 물리학 트리거로 지하실 막 강성을 공부하는 방법

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement ...

연구

암 연구학

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

2D 및 3D 환경에서 신경 전구 세포의 전기장 제어 감독 마이 그 레이션

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

생체공학

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

Compartmentalizing 마이크로 유체 장치와 라이브 세포 이미징을 사용하여 암 줄기 세포 마이그레이션 평가

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

의학

Migratory Behavior of Cells Generated in Ganglionic Eminence Cultures

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral cortex consists of two basic neuronal types: excitatory and inhibitory. These cells arise in distinct areas and migrate into the cortex along different routes (Pearlman et al., '98). Inhibitory interneurons migrate tangentially from subcortical sources, mostly from different regions of the ganglionic eminences (Gelman et al., '09; Xu et al., '04). Their movement requires precise spatiotemporal control imposed by environmental cues, to allow for the establishment of proper cytoarchitecture and connectivity in the cerebral cortex (Caviness &amp; Rakic, '78; Hatten, '90; Rakic, '90). To study the migratory behavior of cells generated in proliferative zones of the ganglionic eminences (GE) in newborn ferrets in vitro we used a 3 dimensional culture arrangement in a BD Matrigel Matrix. The culture setup consisted of two GE explants and a source of tested proteins extracted from the cerebral cortex and adsorbed on fluorescent latex Retrobeads IX positioned between the explants (Hasling et al., '03; Riddle et al., '97). After 2-3 days of culture, the cells start to appear at the edge of the explant showing a propensity to leave the tissue in a radial direction. Live imaging allowed observation of migratory patterns without the necessity of labeling or marking the cells. When exposed to fractions of the protein extract obtained from isochronic ferret cortex, the GE cells displayed different behaviors as judged by quantitative kinetic analysis of individual moving cells.

Time lapse imaging of 3D tissue culture allows studying migratory behavior of individual cells originating from ganglionic eminence in reaction to fractionated protein extract from cerebral cortex.

Ganglionic 에미넨스 문화에서 생성된 세포의 철새 행동

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral ...

신경과학

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

라이브 셀 이미징 분석 실험 연구 세포종 뇌 종양 줄기 세포 이동과 내 습을

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

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

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

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

수정 Zigmond 회의소 분석을 사용하여 트렁크 신경 크레스트 세포 마이그레이션 분석

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

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

미세 채널에서 세포의 이동에 관한 연구

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

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

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

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

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

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

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to extravasate and form a secondary niche.&#160;The extravasation of tumor cells from the blood vascular system can be studied using endothelial cells (ECs) and tumor cells obtained from different cell lines.&#160;Initial studies were conducted using static conditions but it has been well documented that ECs behave differently under physiological flow conditions.&#160;Therefore, different flow chamber assemblies are currently being used to studying cancer cell interactions with ECs.&#160;Current flow chamber assemblies offer reproducible results using either different cell lines or fluid at different shear stress conditions.&#160;However, to observe and study interactions with rare cells such as circulating tumor cells (CTCs), certain changes are required to be made to the conventional flow chamber assembly.&#160;CTCs are a rare cell population among millions of blood cells. Consequently, it is difficult to obtain a pure population of CTCs.&#160;Contamination of CTCs with different types of cells normally found in the circulation is inevitable using present enrichment or depletion techniques.&#160;In the present report, we describe a unique method to fluorescently label circulating prostate cancer cells and study their interactions with ECs in a self-assembled flow chamber system.&#160;This technique can be further applied to observe interactions between prostate CTCs and any protein of interest.

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Our report describes a unique method to visualize and analyze CTC/EC interactions in prostate cancer under physiological flow conditions.

환자와 인간의 내피 세포에서 파생 된 전립선 순환 종양 세포 사이에 E-셀렉틴 매개 상호 작용을 관찰하는 시험 관내 방법에

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

이미징 G의 호중구와 같은 HL60 세포에서 화성과 시그널링 이벤트 수용체가 매개 단백질 결합

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...