플라스미드 Vinculin - GFP는 마이클 데이비슨, 플로리다 주립 대학, 탤러해시, 플로리다의 선물 케네스 야마다, 플라스미드 국립 보건 연구소, 치과와 Craniofacial 연구의 국립 연구소, 베데스다, 메릴랜드, 그리고 굴지 - mRFP에서 선물했다. 이 작품은 NSF 경력 보너스 # 0747334 및 AHA - 국립 SDG # 0835205N Andreea Trache에 의해 지원되었다.

1. 통합 현미경 시스템의 개요 이러한 연구에 사용되는 현미경 시스템은 상세 1에서 설명되었습니다. 간단히, TIRF 첨부 (올림푸스, 센터 밸리, PA)와 거꾸로 올림푸스 IX - 81 현미경과 결합 CSU - 22 요코 스캔 헤드 (요코 전기 병원, 일본). Bioscope SZ 원자 힘 현미경 (Veeco 인스 트루먼 트의 병원, 산타 바바라, CA)는 광학 거꾸로 현미경 위에 탑재하고, 전체 시스템은 연구 등급 광학 테이블의 상단 (뉴 포트, 어빈, CA)에 위치합니다. 회전 디스크 스캐너, 외부 필터 휠과 EMCCD 카메라 (로퍼 과학 - Photometrics, 투싼,​​ AZ)에서 구성된 공촛점 어셈블리가 rigidly 진동 감쇠에 필요한 실리콘 패드에 위치 알루미늄베이스 플레이트와 연결됩니다. 공촛점 스캐너 AFM 측정하는 동안 현미경에서 decoupled 수 treaded 칼라와 플랜지를 통해 현미경으로 연결하는 회전 디스크 스캐너로부터 진동 AFM 측정에 영향을 미치지 않습니다 그러한. 아르곤 - 크립톤 레이저 (스펙트럼 물리학, Mountain View, CA)를 각각 TIRF 및 공촛점 이미징 모드 모두 여기 소스로 사용하고 TIRF 첨부 파일 또는 스캔 머리에 레이저를 전달이 광학 섬유에 또는 결합 수 있습니다 . 두 컴퓨터가 시스템을 제어 : Slidebook 소프트웨어는 광학 이미징을 제어하고 Nanoscope 소프트웨어는 AFM을 제어합니다. 2. 세포 배양 라이브 세포가 특정 단백질에 대한 타겟으로 GFP를 구성 표현은 24 시간 전에 기계적 자극 실험에 유리 바닥 세포 배양 접시에 배치해야합니다. 실험 당일 페놀 레드 무료 매체와 세포 배양 매체를 교체하십시오. 세포 배양 접시는 자기 칼라와 AFM 무대에 장착할 수 있습니다. 3. AFM 팁 준비 및 현미경 설정 2 마이크론과 함께 사용자 정의 AFM 팁 유리 구슬이 DPBS있는 5 번 세탁 후 아비딘 (1 MG / ML)로 5 분 incubated, DPBS 다섯번로 다시 씻어, 다음 1 MG / ML biotinilated 5 분 incubated 것입니다 biotinilated fibronectin. 팁은 끝 2에서 다섯 번 이상 씻어 AFM 스캐너에 탑재하고 보호 실리콘 스커트는 보유자 주위에 배치됩니다 것입니다. 보기의 필드의 중앙에 AFM 팁 정렬에 비디오를 볼 수 있도록 현미경을 설정합니다. 캔틸레버의 가장 끝에있는 레이저 광선을 배치하여 광학 레버를 정렬하고 단일 세포 이미징에 대한 적절한 높은 배율 목표로 목적을 변경합니다. 팁의 상수 봄 실험을 시작하기 전에 결정되어야한다. 이 매개 변수는 Nanoscope 소프트웨어에서 사용 가능한 열 조정 방법을 사용하여 측정됩니다. 개화 이미징 수 있도록 EMCCD 카메라로 비디오 카메라에서 전환합니다. 그런 다음, 셀을 선택하고 특정 세포와 접촉 AFM 팁 가져. 세포 표면과 접촉의 AFM 지점에 양식에 강한 초점 접착에 필요한 20 분 대기 시간 후, 기계적 자극 절차가 시작됩니다. 4. 라이브 세포의 기계 자극 3-4 TIRF 영상은 초점 adhesions를 시각화하고 quantifying을위한 최고의 방법되며, 방적 디스크 공촛점 이미징 5 세포 체내 굴지의 cytoskeleton을 시각화에 사용됩니다. AFM 6 유체 아래의 연락처 모드 이미징에 사용됩니다. 세포의 TIRF 또는 공촛점 이미지가 기계적 자극 절차를 시작하기 전에 취득됩니다. 기계적 자극이 일정 기간 (1-1.5 시간) 동안 매 3-5 분 이산 단계로 상향 이동됩니다 기능화 AFM 프로브에 의해 유발됩니다. 세포 반응 - 반응은 높이 이미지, 1 초 단위 시간을 나타내는 이미지의 각 라인의 시리즈로 Nanoscope 소프트웨어에 지속적으로 기록됩니다. AFM 데이터를 수집하는 동안 작업자는 항상 AFM 팁의 Z 위치는 다음 당기에 대한 적절한 타이밍을 결정하기 위해 모니터링합니다. 각각의 기계적 자극 후 지속 AFM 데이터를 획득하면서 Slidebook 소프트웨어의 세포의 TIRF 또는 공촛점 이미지를 얻을 수 있습니다. 광학 이미지는 굴지의 cytoskeleton과 초점 adhesions의 개편을 보여 영화를 만들 오프라인 처리할 수 있습니다. 또한, 각 이미지는 셀 영역, 특정 단백질 영역을 결정하고, 일반적인 세​​포 형태를 분석하기 위해 양적 처리할 수 있습니다. AFM 팁은 세포와 AFM 팁 감도에서 철회되는 실험의 끝에 Nanoscope 소프트웨어에서 측정됩니다. 실험은 매우 소음 민감합니다! 전 동안 감동 아무 말도 또는 현미경periment은 피해야한다. AFM 측정에서 소음을 최소화하기 위해, 회전 디스크 공촛점는 현미경에서 드 결합해야합니다. 세포 표면과 AFM 팁 사이에 강한 초점 접착력을 형성하는 대기 시간은 사용되는 세포 유형과 끝에 매트릭스 코팅에 따라 달라질 수 있습니다. 5. 대표 결과 <img src="/files/ftp_upload/2072/2072fig1.jpg" alt="그림 1" /> 그림 1. 굴지 - mRFP 및 vinculin - GFP와 transfected 같은 혈관 평활근 세포 (VSMC)가로 몇 군데되었습니다 혀끝의 세포 표면에 (A) AFM, (B) 기저 세포 표면에 TIRF하며, (C) 스피닝 디스크 공촛점 현미경 세포 체내. 공촛점 이미지가 3 차원 스택의 최대 투사로 표시됩니다. AFM과 TIRF 또는 방적 디스크 공촛점 이미지 사이 좋은 중복은 (D)와 (E), 각각에 표시됩니다. 이미지 크기는 64 X 50 미크론이다. <img src="/files/ftp_upload/2072/2072fig2.jpg" alt="그림 2" /> 그림 2. 굴지 - mRFP와 transfected VSMC는 기계 AFM에 의해 자극과 회전 디스크 공촛점 현미경으로 몇 군데했다 화살표 그 굴지의 섬유를 나타냅니다. (A) 진하게 / 중합, (B) depolymerize / 사라, 또는 (c) 때문에 함께 번들 굴지의 구조 조정합니다. 화이트 점선 라인은 세포 위에 AFM 팁을 나타냅니다. 이미지 크기 91 X 91 미크론.

<ol>
	<li>Trache, A., Lim, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+microscopy+for+real-time+imaging+of+mechanotransduction+studies+in+live+cells.">Integrated microscopy for real-time imaging of mechanotransduction studies in live cells.</a> J Biomed Opt. 14, 034024-03 (2009).</li><li>Trache, A., Meininger, G. A., Coico, R., Kowalik, T., Quarles, J. M., Stevenson, B., Taylor, 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=Atomic+Force+Microscopy.">Atomic Force Microscopy.</a> Current Protocols in Microbiology. , 2.1-2.17 (2008).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+imaging+of+surface+fluorescence+with+very+high+aperture+microscope+objectives.">Selective imaging of surface fluorescence with very high aperture microscope objectives.</a> J Biomed Opt. 6, 6-13 (2001).</li><li>Trache, A., Meininger, G. A., Coico, R., Kowalik, T., Quarles, J. M., Stevenson, B., Taylor, 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=Total+Internal+Reflection+Fluorescence+Microscopy.">Total Internal Reflection Fluorescence Microscopy.</a> Current Protocols in Microbiology. , 2.1-2.22 (2008).</li><li>Inoue, S., Inoue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-view+high-speed+confocal+scanner:+the+CSU-10.">Direct-view high-speed confocal scanner: the CSU-10.</a> Methods Cell Biol. 70, 87-127 (2002).</li><li>Binnig, G., Quate, C. F., Gerber, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscope.">Atomic force microscope.</a> Phys Rev Lett. 56, 930-933 (1986).</li><li>Stamenovi&#263;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+mechanics+in+airway+smooth+muscle+cells.">Cytoskeletal mechanics in airway smooth muscle cells.</a> Respir Physiol Neurobiol. 163, 25-32 (2008).</li><li>Gunst, S. J., Tang, D. D., Opazo Saez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+remodeling+of+the+airway+smooth+muscle+cell:+a+mechanism+for+adaptation+to+mechanical+forces+in+the+lung.">Cytoskeletal remodeling of the airway smooth muscle cell: a mechanism for adaptation to mechanical forces in the lung.</a> Respir Physiol Neurobiol. 137, 151-168 (2003).</li><li>Worth, N. F., Rolfe, B. E., Song, J., Campbell, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+smooth+muscle+cell+phenotypic+modulation+in+culture+is+associated+with+reorganization+of+contractile+and+cytoskeletal+proteins.">Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganization of contractile and cytoskeletal proteins.</a> Cell Motility and the Cytoskeleton. 49, 130-145 (2001).</li><li>Romer, H. R., Birukov, K. G., Garcia, J. G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+Adhesions:+paradigm+for+a+signaling+nexus.">Focal Adhesions: paradigm for a signaling nexus.</a> Circulation Research. 98, 606-616 (2006).</li><li>Opazo Saez, A., Zhang, W., Wu, Y., Turner, C. E., Tang, D. D., Gunst, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tension+development+during+contractile+stimulation+of+smooth+muscle+requires+recruitment+of+paxillin+and+vinculin+to+the+membrane.">Tension development during contractile stimulation of smooth muscle requires recruitment of paxillin and vinculin to the membrane.</a> Am J Physiol Cell Physiol. 286, C433-C447 (2004).</li><li>Alenghat, F. J., Ingber, 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=Mechanotransduction:+all+signals+point+to+cytoskeleton,+matrix+and+integrins.">Mechanotransduction: all signals point to cytoskeleton, matrix and integrins.</a> Sciences STKE. 119, pe6-pe6 (2002).</li><li>Zamir, E., Geiger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+complexity+and+dynamics+of+cell-matrix+adhesions.">Molecular complexity and dynamics of cell-matrix adhesions.</a> J Cell Sci. 114, 3583-3590 (2001).</li><li>Geiger, B., Bershdsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+mechanosensory+function+of+focal+contacts.">Assembly and mechanosensory function of focal contacts.</a> Curr Opin Cell Biol. 13, 584-592 (2001).</li><li>Sastry, S. K., Burridge, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+Adhesions:+a+nexus+for+intracellular+signaling+and+cytoskeletal+dynamics.">Focal Adhesions: a nexus for intracellular signaling and cytoskeletal dynamics.</a> Exp Cell Res. 261, 25-36 (2000).</li><li>Orr, A. W., Helmke, B. P., Blackman, B. R., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+mechanotransduction.">Mechanisms of mechanotransduction.</a> Dev Cell. 10, 11-20 (2006).</li></ol>

관심 없음 충돌 선언하지 않습니다.

Cytoskeleton 70-10과 초점 adhesions가 11-16, 조립 분산 및 세포가 마이 그 레이션 또는 기계적 강제로 17 답변으로 넘겨 역동적인 구조입니다. 그들은 세포 성장, 생존, 그리고 유전자 발현을 조절에 중요​​한 기능을합니다. 그들의 구체적인 유통 및 역학을 아는 것은 세포가 모체와 자신 사이의 의사 소통 방법에 대한 이해를 제공합니다. 이 새로운 현미경...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>AFM tip</td>
<td>&nbsp;</td>
<td>Novascan Technologies</td>
<td>&nbsp;</td>
<td>2 micron Borosilicate bead, 0.01N/m, Biotin Surface</td>
</tr>
<tr>
<td>avidin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>A9275</td>
<td>1mg/mL in DPBS</td>
</tr>
<tr>
<td>Fibronectin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>F4759</td>
<td>1 mg/mL</td>
</tr>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21600-051</td>
<td>&nbsp;</td>
<tr>
<td>Amaxa transfection kit for Smooth Muscle Cells</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>VPI-1004</td>
<td>&nbsp;</td>
<tr>
<td>Mattek glass bottom dishes</td>
<td>&nbsp;</td>
<td>MatTek Corporation</td>
<td>P60G-1.5-30-F</td>
<td>Sterile 60mm Falcon dishes with glass bottom coverslip no. 1.5</td>
</tr>		</tbody>
		</table>
		</div>

live cell response to mechanical stimulation studied by integrated optical and atomic force microscopy

살아있는 세포가 기계적 힘을 감지하는 메커니즘을 이해하기 위해서는, 높은 공간과 시간적 해상도를 가진 하위 세포 수준에서 세포의 행동을 조사할 수있는 그들이 반응하고 환경에 적응하는 방법, 새로운 기술하는 것은 개발되었습니다. 따라서, 원자 힘 현미경 (AFM)은 총 내부 반사 형광 (TIRF) 현미경과 고속 회전하는 디스크 (FSD) 공촛점 현미경과 통합되었습니다. 통합 시스템은 실시간으로 기계 자극에 세포 반응의 직접적인 광학 이미징을 허용, 모든 자기편 라이브 세포에서 분자 역동적인 연구 광범 광범위하게 적용됩니다. 굴지의 필라멘트와 초점 adhesions의 중요한 다시 정리하기는 인해 세포 체내 세포 구조로 변화를 유도하는 혀끝의 세포 표면에서 로컬 기계 자극을 표시했다. 이러한 혁신 기술은 기계적 강제에 대한 응답으로 사는 셀 구조 조정과 역학을 이해하는 새로운 정보를 제공합니다. 상세한 프로토콜과 대리인 데이터는 기계적 자극에 표시 라이브 셀 응답이 제공되는 설정합니다.

Watch this Scientific Journal Video about Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy at JoVE.com

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

본 논문은 문화에 살고있는 세포의 기계적 자극에 대한 통합 원자의 운영 독자 강제 광학 이미징 현미경을 지시하는 것을 목표로하고있다. 단계별 프로토콜이 제공됩니다. 기계적 자극에 살고 세포 응답을 보여주는 대표적인 데이터 집합이 제공됩니다.

기계 자극에 라이브 세포 응답 통합 광학과 원자 힘 현미경에 의해 연구

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

live-cell-response-to-mechanical-stimulation-studied-integrated

Research

JoVE Journal

Biology

11.2K 조회수.  Texas A&M Health Science Center. 본 논문은 문화에 살고있는 세포의 기계적 자극에 대한 통합 원자의 운영 독자 강제 광학 이미징 현미경을 지시하는 것을 목표로하고있다. 단계별 프로토콜이 제공됩니다. 기계적 자극에 살고 세포 응답을 보여주는 대표적인 데이터 집합이 제공됩니다.

비디오: Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

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

Phase Contrast and Differential Interference Contrast DIC Microscopy

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

단계 대비 및 차등 간섭 대비 (DIC) 현미경

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

연구

생물학

Presynaptically Silent Synapses Studied with Light Microscopy

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise damaging insults from becoming neurotoxic.  We have been studying a form of presynaptic plasticity that is interesting in part because it is expressed as a digital switching on and off of a presynaptic terminal s ability to release vesicles containing the neurotransmitter glutamate.  Here we demonstrate a protocol for visualizing the activity status of presynaptic terminals in dissociated cell cultures prepared from the rodent hippocampus.  The method relies on detecting active synapses using staining with a fixable form of the styryl dye FM1-43, commonly used to label synaptic vesicles.  This staining profile is compared with immunostaining of the same terminals with an antibody directed against the vesicular glutamate transporter 1 (vGluT-1), a stain designed to label all glutamate synapses regardless of activation status.   We find that depolarizing stimuli induce presynaptic silencing.  The population of synapses that is silent under baseline conditions can be activated by prolonged electrical silencing or by activation of cAMP signaling pathways.

Glutamatergic synapses can switch from an active mode to a silent mode. We demonstrate that presynaptic activity status in dissociated culture of rodent neurons is visualized using a fixable form of the FM1-43 dye to visualize active synapses and immunostaining with vGluT-1 antibody to visualize all glutamate synapses.

라이트 현미경과 연구 Presynaptically 사일런트 시냅스

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

원자 힘 현미경을 사용하여 폐 조직의 미세 기계적 특성

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

단일 비리온 원자력 현미경 검사및 초분해성 형광 이미징을 위한 샘플 준비

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

의 강성 측정<em&gt; 전의 VIVO</em원자 힘 현미경을 사용하여&gt; 마우스 대동맥

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

자석 구슬을 사용하여 기계 인장에 대한 응답으로 세포 표면 접착 리모델링 분석

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

포도당 섭취량 측정 및 인슐린 자극에 대한 반응<em&gt; In Vitro</em&gt; 배양 된 사람의 주요 근육 세포

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Dictyostelium discoideum 응답 심각한 기계적인 자극의 평가

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.

Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.

돌출 힘 현미경 검사 법: 셀 돌출에 의해 개발 된 세력을 계량 하는 방법

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

원자력 현미경을 사용한 살아있는 식물 장기의 일차 세포벽의 기계적 특성 특성 분석

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...