Name: a functional assay for gap junctional examination; electroporation of adherent cells on indium-tin oxide
Uploaded: 2014-10-18T15:00:00.000+00:00
Duration: 11 min 2 s
Description: Watch this Scientific Journal Video about A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide at JoVE.com

We thank Lowell Cochran for expert videography assistance. The financial assistance of the Canadian Institutes of Health Research (CIHR), the Canadian Breast Cancer Foundation (CBCF, Ontario Chapter), the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Breast Cancer Research Alliance, the Ontario Centers of Excellence, the Breast Cancer Action Kingston and the Clare Nelson bequest fund through grants to LR is gratefully acknowledged. SG was the recipient of an NSERC studentship. MG was supported by a postdoctoral fellowship from the US Army Breast Cancer Program, the Ministry of Research and Innovation of the Province of Ontario and the Advisory Research Committee of Queen&#8217;s University.

1 일렉트로 챔버에서 셀 도금 <ol><li> 층류 흐름 후드에서 평소와 같이 멸균 기술을 이용하여 세포를 Trypsinize. 참고 : 그들은 유리에 세포의 확산 방해 할 수 있으므로, 원심 분리하여있는 adherens와 갭 접합 따라서 형성을 트립신의 모든 흔적을 제거하는 것이 매우 중요합니다. </li><li> 피펫 1 (37)의 출력 C 시츄 electroporator에서 (도 1), 및 장소, CO 2 인큐베이터 구비 일렉트로 멸균 챔버에 세포 현탁액의 ML. NOTE : 세포 부착은 피브로넥틴, 콜라겐, 폴리 라이신 또는 세포 및 조직 접착제 (원료 표 참조)에 도금에 의해 개선 될 수있다. </li><li> 세포가 합류 층을 형성 할 때 그들은 GJIC 시험에 대한 준비가되어 있습니다. </li></ol> 2 일렉트로 절차 GJIC 검사 용 전기 천공은 층류 흐름 후드 외부에서 수행 될 수있다이 걸리기 때문에 단 몇 분. 긴 배양 시간은 특정 실험에 요구된다면, 그것은 층류 흐름 후드에서 전적으로 수행 될 수있다. 모든 경우에서, 세포는 성장 챔버는 멸균해야한다. <ol><li> 5 ㎎ / ㎖ 루시퍼 노란색 솔루션을 준비 : 2 ㎖의 칼슘이없는 성장 매체 (electroporator 제공) 10 mg을 LY 분말을 녹여. 4 O C.에서 용액 저장을 필터 멸균 긴 배양 시간을 필요로 실험 </li><li> 성장 매체를 기음과 단층을 긁거나 세포를 건조하지 않도록주의, 칼슘이없는 배지로 세포를 씻어. 세포가 건조하면 그들은 일반적으로 어두운 핵을 가지고 일렉트로없이 LY를 선택합니다. 효과는 더욱 공기 드래프트 (도 4F)에 노출되는 슬라이드의 중간에 더 현저하다. </li><li> 에펜 도르프 피펫으로, 챔버 (B)의 에지에서, 셀 (챔버 전체를 400 μL)에 염료 용액을 피펫주의 eing입니다 것은 셀 층을 만지지 않도록. </li><li> electroporator와 함께 제공되는 홀더에 실을 배치하고 적절한 강도 (토론 참조)의 펄스를 적용합니다. </li><li> 조심스럽게 LY 솔루션의 일부를 대기음. 덜 중요한 실험 번 재사용 될 수있다. </li><li> 10 % 투석 혈청을 포함하는 칼슘이없는 DMEM를 추가합니다. </li><li> 갭 정션을 통한 염료 전달​​을 허용하도록 배양기에서 3-5 분 동안 세포를 배양한다. 참고 :이 시점에서 투석 혈청의 포함은 기공 폐쇄를하는 데 도움이됩니다. </li><li> 라이브 세포 관찰을위한 칼슘이없는 DMEM으로 통합되지 않은 염료를 세척 할 것. 대안 적으로, 세포를 웰에 4 % 포름 알데히드의 첨가를 통해,이 단계에서 고정 될 수 있고, 그 다음 PBS (인산염 완충 식염수)로 세척 하였다. 모든 경우에 세척 모든 배경을 제거해야합니다. 주 : 예비 실험이 전압이 너무 낮은 경우, 최적의 조건을 결정하는 데있어서, 그것은 세포 incubato 회복하도록 할 수있다몇 분을위한 연구 및 슬라이드의 비용을 절약하기 위해 같은 슬라이드를 두 번 electroporate. </li></ol> 3 현미경 검사 <ol><li> 사용되는 염료에 대한 적절한 필터를 구비 한 반전 된 현미경을 사용하여, 형광 조명 하에서 세포를 관찰한다. 노란색 루시퍼를 들어, 여자는 423nm, 방출 555nm, 니콘 IX70 현미경에 대한 WBV 필터입니다. </li><li> 위상차 하에서 세포 형태를 조사하기 위해, 액체와 함께 상부에 충전 한 후, 잘 플라스틱 위에 유리 커버 슬립을 배치하여 메 니스 커스의 효과를 제거한다. 좋은 사진 촬영을 위해, 웰 슬라이드에서 분리 될 수 있으며, 커버 슬립은 프레임에 배치. 생균 대를 성장 배지로 채워 져야하는 동안 고정 된 세포의 경우, 웰, 현미경 관찰 용 글리세롤로 채워질 수있다. 참고 : 세포가 염료 (위의 단계 2.7)의 전송 다음 포름 알데히드로 고정하는 경우 세탁 촬영이 용이하다. 세포가있는 경우 더t는 형광 고정 세포 형광 몇 시간 동안 유지가 도입하면서 LY의 세포 유형, 전압 및 양에 따라 약 60 분 이내에 세포로부터 제거하고, 고정. 그러나, 형광 페이드 또는 고정 된 세포에서, O / N 배양 후 소산. 이러한 이유로, 사진은 일렉트로 (그림 2) 직후에주의해야합니다. </li></ol> 세포 간 통신의 4 정량 <ol><li> 형광 및 위상 대비 (그림 3)에서 20 배 목적으로 세포를 사진. </li><li> 비 electroporation하여 영역 (그림 2, 화살표, 일렉트로 에지)과 경계에 electroporation하여 세포를 확인하고 별표 표시합니다. </li><li> 염료가 갭 접합을 통해 전송 한 비 electroporation하여 측면에 형광 세포를 확인하고 점으로 표시 (그림 3a 및 3b). </li><li> 의 총 수를 나눈다에지 (도 3a 및 3b)을 따라 electroporation하여 세포의 수에 의해 비 전기 천공 영역에 세포를 형광. NOTE 적어도 200 연속 electroporation하여 경계 셀로부터의 전송을 각각의 실험에 대해 계산된다. 얻어진 숫자는 GJIC 값이다. </li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+inhibition+of+Growth+Factor-stimulated+ERK1/2+activation+in+intact+cells+by+electroporation+of+a+Grb2-SH2+binding+peptide.">Specific inhibition of Growth Factor-stimulated ERK1/2 activation in intact cells by electroporation of a Grb2-SH2 binding peptide.</a> Cell Growth Differ. 11, 293-303 (2000).</li><li>Meda, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+the+molecular+permeability+of+connexin+channels.">Assaying the molecular permeability of connexin channels.</a> Methods Mol Biol. 154, 201-224 (2001).</li><li>Hofgaard, J. P., Mollerup, S., Holstein-Rathlou, N. H., Nielsen, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+gap+junctional+intercellular+communication+based+on+digital+image+analysis.">Quantification of gap junctional intercellular communication based on digital image analysis.</a> Am J Physiol Regul Integr Comp Physiol. 297 (2), R243-R247 (2009).</li><li>Raptis, L., Lamfrom, H., Benjamin, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cellular+phenotype+and+expression+of+polyomavirus+middle+T+antigen+in+rat+fibroblasts.">Regulation of cellular phenotype and expression of polyomavirus middle T antigen in rat fibroblasts.</a> Mol Cell Biol. 5, 2476-2486 (1985).</li><li>Raptis, L., Marcellus, R. C., Whitfield, 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=High+membrane-associated+protein+kinase+C+activity+correlates+to+tumorigenicity+but+not+anchorage-independence+in+a+clone+of+mouse+NIH+3T3+cells.">High membrane-associated protein kinase C activity correlates to tumorigenicity but not anchorage-independence in a clone of mouse NIH 3T3 cells.</a> Exp Cell Res. 207, 152-154 (1993).</li><li>Vultur, A., Cao, J., Arulanandam, R., Turkson, J., Jove, R., Greer, P., Craig, A., Elliott, B. E., Raptis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+to+cell+adhesion+modulates+Stat3+activity+in+normal+and+breast+carcinoma+cells.">Cell to cell adhesion modulates Stat3 activity in normal and breast carcinoma cells.</a> Oncogene. 23, 2600-2616 (2004).</li><li>Vultur, A., Arulanandam, R., Turkson, J., Niu, G., Jove, R., Raptis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stat3+is+required+for+full+neoplastic+transformation+by+the+Simian+Virus+40+Large+Tumor+antigen.">Stat3 is required for full neoplastic transformation by the Simian Virus 40 Large Tumor antigen.</a> Mol Biol Cell. 16, 3832-3846 (2005).</li></ol>

The corresponding author is the inventor in a patent held by Queen&#8217;s University, which has been licensed to Cell Projects Inc. The company or the University had no influence upon the contents of the paper.

프로토콜의 중요한 단계 electroporation하여 재료 전기 천공 할 물질의 순도는 매우 중요하다. 세포가 매우 평탄한 경우 추적 염료의 다음 높은 농도 (20 mg을 LY 대 / ㎖까지)와 같은 경우에 사용되어야하며, 순도가 더욱 중요 더 구형 1 세포에서보다. LY 외에 다른 염료 또는 nonpermeant 분자의 많은 종류가 상이한 connexins 이루어진 25 채널 프로브로 사...

셀 전류의 애플리케이션 프로세스라고 일렉트로, 세포막에 구멍을 형성시킨다. 기공이 막을 통한 분자의 nonpermeant 다양한 통로를 허용한다. 전기장이 형성된 기공은 매우 작고 휴대 생리학 방해를 최소화하여, 급속 리 클로즈되도록 정확하게 제어 할 수있다. 흥미롭게도, 부착 세포는 현탁액에 전기 천공 할 분리되지 않고, 그들이 성장 표면에 도전성 투명 인듐 - 주석 산화물 (ITO)로 코팅하고, 반응계에서 electroporation하여 유리 슬라이드 상에 성장 될 수있다. 세포는이 표면 상에 잘 성장할 수 있으며, 이들이 결합하고 확장 한, 상세한 현미경 관찰이 가능하다. 이 기술을 사용하여, 소형 분자 nonpermeant 즉시 도입하고 미리보기의 활성화에 대한 연구를 위해 적합하게이 기술을 세포의 본질적으로 100 %로 할 수있다(1 검토) 수용체의 리간드 자극 다음과 같은 경로의의 onents. 일렉트로는 DNA의 도입 (또한 electrotransfection)에 주로 사용되어왔다. 그러나 현장에서 일렉트로는 전사 인자 바인딩, 또는 siRNA를 3,5 억제하는 등의 안티센스 RNA, 이중 가닥 DNA 미끼 올리고 뉴클레오티드 등의 펩티드 2-4, 올리고 뉴클레오티드 분자의 큰 다양성의 도입을 위해 도움이 될 수 있습니다, 6, 방사성 뉴클레오티드 7-10, 단백질 11 (12) 또는 전구 약물 13. 일렉트로 후, 세포는 생화학 적 분석을 위해 용해 또는 고정 및 항체로 염색 할 수 있습니다. 셀이 전도성 코팅의 가장자리에 걸쳐 증가함에 따라 세포의 성장이,이 표면의 높이 차이에 의해 방해되지 않도록 도전성 ITO 코팅은 800-1,000Å 매우 얇다. 이 장점이 아닌 electrop을 제공합니다orated 세포는 제어 역할을하는, electroporation하여 사람과 나란히 성장시킬 수있다. 비디오에서 설명한 것과 동일한 접근은 갭 접합부, 세포 간 통신 (GJIC)의 검사를 위해 사용될 수있다. 갭 접합 인접 셀 (14)의 내부를 연결하는 채널이다. 이러한에서 Src와 같은 종양 유전자가 GJIC (15, 16)를 억제하면서 갭 접합부는, 세포 간 통신은, 종양 형성 및 전이에 중요한 역할을한다. 루시퍼 옐로우 (LY) 같은 미세 주입을 통해 배양 된 세포에 도입하거나 긁어로드 (17)와 인접 셀에 염료의 확산이 현미경 형광 조명 아래에서 관찰 등의 GJIC에게 형광 염료를 검사합니다. 그러나 이러한 기술은 변함없이 세포 손상을 야기한다. 이제 셀 (18)에 부분적으로 ITO로 코팅 된 유리 슬라이드 상에 성장되는 기법을 설명한다. 전기 펄스 LY (또는 다른 염료)의 존재 캘리포니아 도포인접 비 electroporation하여 세포에 염료 이행는 형광 현미경을 통해 관찰 조명하면서, 상기 슬라이드의 도전 부에 성장 세포로의 침투를 사용. 단일 층으로부터 분리하는 경향이있다 감수성 세포 교란을 방지하기 위해, 전극을 필요로하지 않은 설계되었다 조립체는 전류 (19)를 적용하는 셀의 상단에 배치된다. 혈청 자극 (12) 다음의 G1 단계의 길이에 대한 효과의 부재에 의해 표시된 바와 같이이 방법은 FOS의 수준 증가, 세포 대사에 어떤 검출 가능한 방해하지 않고, 다수의 셀에서 GJIC을 정량 할 수있는 능력을 구비 protooncogene 단백질 (Raptis, 미 출판) 또는 셀룰러 스트레스, P38 돼지 또는 JNK / SAPK 키나제 1과 관련이 키나아제. 종양 유전자 발현 변화와 GJ의 레벨 사이의 링크의 시험이 가능하게 접근IC (18)뿐만 아니라, 폐 종양 표본 20-23 갓 배양 된 세포를 포함하여 다양한 종류의 세포에서 GJIC시에서 Src와 STAT3의 효과. 또한, 기판에 대한 세포 부착은 그 단계에서 환원 19,24되지만, 성공적 adipocytic 분화시 갭 정션 폐쇄 시연에 이용 된 상부 전극을 결여 한 바와 셋업과 동일계에서 일렉트로.

<table><tbody><tr><td>DMEM</td><td>Cellgro http://www.cellgro.com/</td><td>50-013-PB</td><td></td></tr><tr><td>DMEM without Calcium</td><td>Hyclone (Thermo scientific: http://www.thermoscientific.com)</td><td>SH30319-01</td><td></td></tr><tr><td>Donor Calf Serum&amp;nbsp;</td><td>PAA: http://www.paa.com/</td><td>Cat.# B15-008</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>PAA: http://www.paa.com/</td><td>Cat.# A15-751</td><td></td></tr><tr><td>Electroporation apparatus</td><td>Cell Projects Ltd UK: http://www.cellprojects.com/</td><td>ACE-100</td><td></td></tr><tr><td>Chambers</td><td>Cell Projects Ltd UK</td><td>ACE-04-CC</td><td>4-wells</td></tr><tr><td>Chambers</td><td>Cell Projects Ltd UK</td><td>ACE-08-CC</td><td>8-wells</td></tr><tr><td>Lucifer Yellow</td><td>Cell Projects Ltd UK</td><td>ACE-25-LY</td><td>High purity</td></tr><tr><td>CelTak</td><td>BD Biosciences</td><td>354240</td><td>Cell and tissue adhesive</td></tr><tr><td>Fibronectin</td><td>Sigma Aldrich</td><td>F1141</td><td></td></tr><tr><td>Collagen</td><td>BD Biosciences</td><td>354236</td><td></td></tr><tr><td>Poly-Lysine</td><td>Sigma Aldrich</td><td>P8920</td><td></td></tr></tbody></table>

a functional assay for gap junctional examination; electroporation of adherent cells on indium-tin oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

그림 2는 고정 및 세탁 다음과 같은 형광 (패널 A와 B)에서 LY와 전기 천공 및 촬영 쥐 간 상피 T51B 셀 (22), 또는 위상차 (C) 조명을 보여줍니다. 패널에 electroporation하여 영역의 가장자리가 빨간색으로 표시됩니다. 적색 라인의 오른쪽에 형광 구배 갭 정션을 통한 전송을 나타낸다. 염료가 전송 한 셀이 도트로 표시된 상태에서, 전기 천공 영역의 에지에서 셀 스타 식별되고 표시되...

Watch this Scientific Journal Video about A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide at JoVE.com

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

갭 접합부 시험에 대한 기능 분석; 인듐 - 주석 산화물에 자기편 세포의 전기 천공 법 (electroporation)

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

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JoVE Journal

Biology

9.9K 조회수.  Queen's University. This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

비디오: A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

에 대한 접착 주파수 분석 현장에서</em셀 - 셀 인터페이스에서 교차 Junctional 분자의 상호 작용> 속도론 해석

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

연구

생체공학

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

개발<em&gt; 체외</em&gt; 분석은 중간 엽 세포를 시행 한 상피 - 중간 엽 전환의 수축 기능을 평가하는

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

발생학

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 ...

신경과학

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

이온 채널의 돌연변이 유발과 기능 분석 Heterologously 포유 동물 세포에 표현

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

생물학

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

표현과 HEK - 293T 세포의 재조합 전압 - 게이 티드 이온 채널 Electrophysiological 녹음에 최적화된 Transfection 전략

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

휴대 통신 분석을위한 단일 세포 미세 주입

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

<meta charset="utf8"></head><body>A431 세포의 전기천공법 분석 및 전달 예측을 위한 TEEI 방법

Porous Substrate-Based Electroporation with Transepithelial Electrical Impedance Monitoring

Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other types of intracellular delivery, PSEP relies heavily on fluorescent markers and fluorescent microscopy to determine successful delivery. To gain insight into the intermediate steps of the electroporation process, a PSEP platform with integrated transepithelial electrical impedance (TEEI) monitoring was developed. Cells are cultured in commercially available inserts with porous membranes. After a 12 h incubation period to allow for the formation of a fully confluent cell monolayer, the inserts are placed in transfection media located in the wells of the PSEP device. The cell monolayers are then subjected to a user-defined waveform, and delivery efficiency is confirmed through fluorescent microscopy. This workflow can be significantly enhanced with TEEI measurements between pulsing and fluorescent microscopy to collect additional data on the PSEP process, and this additional TEEI data is correlated with delivery metrics such as delivery efficiency and viability. This article describes a protocol for performing PSEP with TEEI measurements.

<meta charset="utf8"></head><body>저자 스포트라이트: 향상된 모니터링 및 중간 단계 특성화를 위해 마이크로 및 나노 채널을 통한 다공성 기판 전기천공법 최적화

Porous substrate electroporation (PSEP) pairs consistent, high throughput delivery with high cell viability. Introduction of transepithelial electrical impedance (TEEI) measurements provides insight into the intermediate processes of PSEP and allows for label-free delivery. This article discusses a method for performing PSEP delivery experiments and TEEI measurement analysis simultaneously.

Transepithelial Electrical Impedance Monitoring을 사용한 다공성 기판 기반 전기천공법

Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

갭 접합부 세포 간 통신 : 천연 제품의 독성 물질과 독소의 반작용을 평가하기위한 기능성 바이오 마커, 보건 혜택

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

갭 접합부 시험에 대한 기능 분석; 인듐 - 주석 산화물에 자기편 세포의 전기 천공 법 (electroporation)

요약

더 많은 비디오 탐색

이 비디오의 챕터

에 대한 접착 주파수 분석 현장에서 속도론 해석

개발

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

이온 채널의 돌연변이 유발과 기능 분석 Heterologously 포유 동물 세포에 표현

표현과 HEK - 293T 세포의 재조합 전압 - 게이 티드 이온 채널 Electrophysiological 녹음에 최적화된 Transfection 전략

휴대 통신 분석을위한 단일 세포 미세 주입

Transepithelial Electrical Impedance Monitoring을 사용한 다공성 기판 기반 전기천공법

Transepithelial Electrical Impedance Monitoring을 사용한 다공성 기판 기반 전기천공법

Transepithelial Electrical Impedance Monitoring을 사용한 다공성 기판 기반 전기천공법

갭 접합부 세포 간 통신 : 천연 제품의 독성 물질과 독소의 반작용을 평가하기위한 기능성 바이오 마커, 보건 혜택