연구는 NIH 보조금 EY012031과 EY0182175 및 FM 커비 재단에 의해 지원되었다.

문화 고립된 세포를 조작을위한 광학 핀셋 광학 핀셋의 트래핑 세력은 빛의 운동량 (.; Ashkin 외, 1986 Ashkin, 1991)에서 생성됩니다. 이러한 세력 쉽게 정지 세포를 함정에 있지만, 그들은 표면에 부착 세포를 이동할 수 없습니다. 따라서 세포가 갇혀있는 시점에서 문화 요리에 부착을 줄이기 위해, coverslip는 구충제 세포와 폴리 - 2 - hydroxyethylmethacrylate (폴리 - HEMA) (시그마 화학 (주), 세인트 루이스, MO), 독성 화합물로 코팅되어 속성은 이전에 내피 세포 (Folkman과 Moscona, 1978)에 접착 연구에 대한 고용. 폴리 - HEMA의 문화 요리 중 하나가 절반을 다루고 있지만, 다른 반쪽은 세포의 성장을 지원하는 기판과 코팅입니다. 저희 연구실에서는 샐 - 1 culturing의 샐러맨더 망막 세포에 대한 기판으로 사용됩니다. 살 - 1 fabricating 및 폴리 - HEMA 코팅 요리가 아래에 설명하는 절차 : 문화 요리 제작 <ol><li> 산이 클린 coverglasses (# 1 VWR 과학 주식 회사, 미디어, PA). 이 coverglass은 레이저를 초점에 필요한 높은 수치 조리개 목적에 필요한 단지 0.1mm의 두께의에 대한 선정되었습니다. </li><li> 셀 repellant 영역을 만들려면, 실온에서 하룻밤 95 % 에탄올의 1ml 20 MG 폴리 - HEMA를 (시그마 화학 (주), 세인트 루이스, MO) 디졸브. 가능한 한,이 솔루션은 준비 1 개월 이내에 사용해야합니다. </li><li> 같은 두건으로 먼지없는 환경에서 거의 수직 위치에서 coverglasses을 프로프. </li><li> 200μl micropipette의 플라스틱 팁에서 coverglass 상단의 폴리 - HEMA 솔루션 중 하나 또는 두 방울을 넣으십시오. 방울 (각 50 - 100μl 정도) coverglass의 절반으로 제한해야합니다. 가파른 경사면은 액체가 폴리 - HEMA의 얇은 코팅도를 제공하고, 아래로 coverglass의 표면 아래로 흐르는 보장합니다. 폴리 - HEMA 코팅 coverglasses는 실온에서 30~60분 위해 후드의 건조하도록 허용합니다. </li><li> 가장자리에 가까운 훌륭한 팁 펜으로 coverglass의 폴리 - HEMA 측면을 표시합니다. </li><li> 35mm 문화 접시의 바닥에 1cm의 구멍을 드릴. </li><li> 접착제 폴리 - HEMA 코팅 쪽이 접시의 안쪽 방향으로 얼굴을 너무 Sylgard 184 (다우 코닝 (주), 미들랜드, MI)와 문화 요리의 하단에 폴리 - HEMA 코팅 coverglass. 폴리 - HEMA 코팅의 가장자리를 확인은 구멍의 중심을 실행합니다. 요리 먼지없는 환경에서 실온에서 1-3일 위해 건조하실 수 있습니다. </li><li> 폴리 - HEMA 코팅의 가장자리 아래의 다이아몬드 팁 펜으로 coverglass의 외부에 라인을 스크래치. 그렇다면, 그것 떨어져 약 2mm에 직각에 두 줄을 건너 스크래치. 교차로의 두 지점이 참조 또는 현미경 단계에 문화 접시 같은 방향을 복제에 사용되는 fiducial 포인트 역할을합니다. </li><li> 야간 자외선 아래에서 요리를 소독. </li><li> 망막 세포 점막 (넉넉한 박사 피터 MacLeish, 의학 모어 하우스 학교에 의해 제공에 대해 제기된 마우스 방지 샐러맨더 항체를 포함하는 문화 요리의 휴대 자기편 반 코트 샐 - 1 이외의 폴리 - HEMA 절반을 만들려면 , 아틀란타, GA;. MacLeish 외 (1983)를 ​​참조). 첫째, 외투 약 100μl 0.1의 MG / ML 살균 염소 안티 - 마우스 IgG 항체 (Boehringer 만하임 공사, 인디애나 폴리스)와 살 - 1 면을, 폴리 - HEMA쪽으로을 통해 액체를 누설하지 않도록주의한다. 세 시간 동안 떠나 다음, 그것을 제거합니다. 한 두 번, 다음 접시의 폴리 - HEMA 측면에 액체를 누설하지 돌보는, 100μl 샐 - 1 뜨는 배치 살균 링어의 솔루션과 같은 영역을 씻으십시오. 첫 번째 항체는 살 - 1 알려진 농도에 코팅되어 보장합니다. </li><li> 살 - 1 하룻밤과 요리를 품다. </li><li> 살 - 1 항체 솔루션을 제거하고 멸균 링어의 솔루션으로 영역을 씻어. 전지 도금으로 직전 접시 2 ML 매체를 소개합니다. </li></ol> 수륙 양용 링어의 솔루션, 혈청없는 양용 매체 및 효소 소화에 대한 링어의 솔루션의 구성은 MacLeish와 타운스 - 앤더슨 (1988)과 멘델 외에서 발견됩니다. (1993). 세포 배양의 준비 이 동영상에 사용되는 세포에서 파생되었다 가벼운 적응, 성인, 수중 상 호랑이 도롱뇽은 (Ambystoma tigrinum), 길이 17~22센티미터 측정 (찰스 설리반 주식 회사, 내쉬빌, TN), 5 유지 ° C를에 12 H : 12 H 빛을 어두운주기. 프로토콜은 기관 애니멀 케어 승인 및 국립 보건원의 지침에 따라 엄격 의약과 뉴저지의 치과 대학위원회 (IACUC)를 사용했다. 망막 세포 문화를 준비하기위한 절차 Nachman - Clewner와 타운스 - 앤더슨 (1996)에 의해 설명 다음과 같이 있습니다 : <ol><li> 동물을 참살하고 속. 눈을 제거하고 각막과 홍채를 해부하다. 망막은 세안 컵 안에 약간 분리해야합니다. 망막 밑에 곡선 포셉를 배치함으로써, 망막을 특종. 렌즈가이 단계에있는 망막에 연결된 경우 그것은 중요하지 않습니다. </li><li> 40 분 대한 샐러맨더 링어의 솔루션에 14 U / ML papain (워싱턴, 프리 홀드, NJ)에서 망막을 소화. </li><li> 사용하기 전에, papain 링어의 솔루션은 30 분 대한 95% O 5분의 2 %의 CO 2 가스로 부풀어 오른해야합니다. 다음, 0.22 미크론 필터 (Millipore, Carrigtwohill, 아일랜드)를 통해 통과 소독. </li><li> 무균 샐러맨더 링어의 솔루션을 세 번있는 망막을 씻어. </li><li> 링어의 솔루션에서 렌즈를 제거하고 부드럽게 세포 현탁액을 양보하는 피펫을 보어 3mm로 망막 조직을 씹다. </li></ol> 절연 뉴런의 레이저 핀셋 조작 <ol><li> 무대에 기준점과 함께 접시에 표시를 정렬하여 요리를 orienting, 현미경 단계에 문화 접시를 놓습니다. 컴퓨터에 fiducial 지점의 좌표를 저장합니다. </li><li> 플레이트 살 - 1 모두에 세포와 요리의 폴리 - HEMA 반쪽. </li><li> 세포는 항체 기판에 연결하는 요리의 점착 성의 측면에 전지 오래되는, 약 20 분에 대한 해결하실 수 있습니다. </li><li> 접시의 살 - 1 점착 성의 측면에서 셀을 선택합니다. x와 y 단계의 세포에 가까운 위치의 좌표를 표시합니다. 우리의 경우에는, 요점은 선택된 셀의 기본 dendrites에서 약 10μm했다. 컴퓨터에 좌표를 저장합니다. </li><li> 접시의 폴리 - HEMA 측면에 우리 사건 photoreceptor에 두 번째 신경 세포를, 선택하고 그것을 함정에 레이저 트위터 도구를 사용하십시오. 트래핑은 전력 수준의 넓은 범위에서 얻을 수 있습니다. 그러나, 우리는 10 % 세포와 함께 트래핑 파편을 피하기 위해 충분히 낮은 -20 %에서 레이저의 전원을 설정하지만, 매체를 통해 세포를 전송하기에 충분한. </li><li> 트랩에서 셀을 누른 상태에서 세포가 잘 문화 접시의 표면 위 및 연결된 뉴런 위에 있도록 무대를 낮춥니다. 살 - 1 측면에 이전에 저장한 x와 y 좌표로 세포를 가지고 컴퓨터를 제어에서 단계를 이동합니다. 무대의 움직임은 8 20μm / s의 설정했지만, 최대 속도는 경험적으로 결정하여야한다. 살 - 1 면에 대상 지점에 도착, 접시의 표면에 갇혀있는 셀을 가져 무대를 올립니다. 셀 배치 몇 미크론의 정밀도로 만들 수 있습니다. 세포가 살 - 1 기판을 준수하도록 허용합니다. </li><li> 쌍 형성 전후 쌍의 두 세포의 디지털 이미지를 취득하는 것은 유용한 기록을 제공합니다. </li><li> 인큐베이터에있는 셀 쌍을 유지합니다. 샐러맨더 세포 들어, 10 humidified 챔버에서 요리를 배치 ° C. 세포 영상 시간 경과에 의해 모니터링하고 필요한 세포 생물 학적 기법과 함께 사용할 수 있습니다. </li></ol>

<ol>
	<li>Ashkin, A., Dziedzic, J. M., Bjorkholm, J. E., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+a+Single-Beam+Gradient+Force+Optical+Trap+for+Dielectric+Particles.">Observation of a Single-Beam Gradient Force Optical Trap for Dielectric Particles.</a> Opt. Lett. 11, 288-290 (1986).</li><li>Clarke, R. J., Hoegnason, K., Brimacombe, M., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cone+and+rod+cells+have+different+target+preferences+in+vitro+as+revealed+by+optical+tweezers.">Cone and rod cells have different target preferences in vitro as revealed by optical tweezers.</a> Mol. Vision. 14, 706-720 (2008).</li><li>Folkman, J., Moscona, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cell+shape+in+growth+control.">Role of cell shape in growth control.</a> Nature. 273, 345-349 (1978).</li><li>Liu, Y., Cheng, D. K., Soneck, G. J., Berns, M. W., Chapman, C. F., Tromberg, B. J. <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+localized+cell+heating+induced+by+infrared+optical+tweezers.">Evidence of localized cell heating induced by infrared optical tweezers.</a> Biophysical Journal. 68, 2137-2144 (1995).</li><li>MacLeish, P. R., Barnstable, C. J., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+monoclonal+antibody+as+a+substrate+for+mature+neurons+in+vitro.">Use of a monoclonal antibody as a substrate for mature neurons in vitro.</a> Proc. Natl. Acad. Sci. USA. 80, 7014-7018 (1983).</li><li>MacLeish, P. R., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+synapse+formation+among+major+classes+of+adult+salamander+retinal+neurons+in+vitro.">Growth and synapse formation among major classes of adult salamander retinal neurons in vitro.</a> Neuron. 1, 751-760 (1988).</li><li>Mandell, J. W., MacLeish, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Townes-Anderson+E.+Process+outgrowth+and+synaptic+varicosity+formation+by+adult+photoreceptors+in+vitro.">Townes-Anderson E. Process outgrowth and synaptic varicosity formation by adult photoreceptors in vitro.</a> J. Neurosci. 13, 3533-3548 (1993).</li><li>Nachman-Clewner, M., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injury-induced+remodelling+and+regeneration+of+the+ribbon+presynaptic+terminal+in+vitro.">Injury-induced remodelling and regeneration of the ribbon presynaptic terminal in vitro.</a> J. Neurocytol. 25, 597-613 (1996).</li><li>Townes-Anderson, E., St Jules, R. S., Sherry, D. M., Lichtenberger, J., Hassanain, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromanipulation+of+retinal+neurons+by+optical+tweezers.">Micromanipulation of retinal neurons by optical tweezers.</a> Mol. Vis.. 4, (1998).</li></ol>

라이트 추진력을 가지고 있으며, 그것이 세포를 통해 전달과 같은 광선이 굴절 때 힘이 추진력의 방향을 변경해야합니다. 때문에 운동량 보존의 법칙의 반대 방향으로 포스가 차례로, 휴대폰으로 다시 반응해야합니다. Ashkin (1991)은 현미경의 목적 렌즈의 초점 레이저 빔을에 의해 생성되는 힘은 초점의 중심 방향으로 셀 이동합니다 것으로 나타났다. 레이저 광선의 힘을 몇 piconewtons를 생성하더라도,이 힘을는 매...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<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>25mm circle No.1 coverglass</td>
<td>&nbsp;</td>
<td>VWR Scientific Inc., Westchester, PA</td>
<td>48380 080</td>
<td>&nbsp;</td>
<tr>
<td>poly-2-hydroxyethylmethacrylate (poly-HEMA)</td>
<td>&nbsp;</td>
<td>Sigma Chemical Co., St Louis, MO</td>
<td>P-3932</td>
<td>Dissolve in 95% ethanol</td>
</tr>
<tr>
<td>Goat anti-mouse IgG antibody</td>
<td>&nbsp;</td>
<td>Chemicon International, Temecula CA</td>
<td>AP181</td>
<td> 1mg in 1ml, dilute 10x for use</td>
</tr>
<tr>
<td>Sal-1 supernatant containing mouse anti-salamander antibody</td>
<td>&nbsp;</td>
<td>generously provided by Dr. Peter MacLeish</td>
<td>&nbsp;</td>
<td>Dr. Peter MacLeish, Morehouse School of Medicine, Atlanta, GA</td>
</tr>
<tr>
<td>3 mm bore 5ml pyrex disposable pipets</td>
<td>&nbsp;</td>
<td>Corning Inc., Corning NY</td>
<td>7078A-5</td>
<td>&nbsp;</td>
<tr>
<td>Cell culture dishes 35mm x 10mm</td>
<td>&nbsp;</td>
<td>Corning inc., Corning NY</td>
<td>430165</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 silicone elastomer kit</td>
<td>&nbsp;</td>
<td>Dow Corning Corp., Midland MI</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Optical tweezers-microtool or laser tweezers</td>
<td>&nbsp;</td>
<td>Cell Robotics Inc., Albuquerque NM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1 W continuous wave diode laser of 980nm wavelength</td>
<td>&nbsp;</td>
<td>Cell Robotics Inc., Albuquerque NM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Axiovert 100 inverted light microscope</td>
<td>&nbsp;</td>
<td>Carl Zeiss Inc., Thornwood, NY</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>40x oil immersion plan neofluor objective lens</td>
<td>&nbsp;</td>
<td>Carl Zeiss Inc., Thornwood, NY</td>
<td>&nbsp;</td>
<td>Numerical aperture (N.A. 1.3)</td>
</tr>
<tr>
<td>Black and white CCD camera</td>
<td>&nbsp;</td>
<td>Sony Corporation, Tokyo, Japan</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Computer and joystick with software</td>
<td>&nbsp;</td>
<td>Cell Robotics Inc.</td>
<td>&nbsp;</td>
<td>for controlling a motorized stage</td>
</tr>		</tbody>
		</table>
		</div>

using laser tweezers for manipulating isolated neurons in vitro

본 논문 및 비디오에서 우리는 체외에서 성인 망막 뉴런의 재생 세포 프로세스의 타겟팅 설정을 연구하기 위해 실험실에서 사용되는 프로토콜을 설명합니다. 망막 세포 문화를 준비 절차, 특히 레이저 핀셋 사용하기 위해 만든 요리에 고립 망막 세포의 도금과 절개, 소화 및 망막의 분쇄 및 최종 함께 시작합니다. 이들 요리는 세포 접착제의 절반과 세포 repellant 절반으로 나누어집니다. 세포 접착제 사이드는 우리의 세포가 성장하는시 기판을 제공 살 - 1 항체의 레이어로 코팅됩니다. 기타 접착제 기판은 다른 세포 유형에 사용할 수 있습니다. 셀 repellant 사이드는 폴리 - HEMA의 얇은 레이어로 코팅됩니다. 접시의 폴리 - HEMA 측면에 도금 세포가 레이저 핀셋과 함께 함정에 수송 그리고 쌍을 만들기 위해 살 - 1 면에 세포에 인접 배치됩니다. 어떤 크기의 셀 그룹의 형성이 기술이 가능해야합니다. &quot;레이저 핀셋 제어 미세 조작은&quot;조사가 세포가 이동을 선택할 수있다는 것을 의미하고, 세포 사이의 원하는 거리는 표준화 수 있습니다. 레이저 빔은 문화 요리의 투명한 표면을 통해 이뤄지 때문에, 세포의 선택과 게재는 동봉된, 무균 환경에서 수행하고 있습니다. 세포 영상 시간 경과에 의해 모니터링하고 필요한 세포 생물 학적 기법과 함께 사용할 수 있습니다. 이 기술은 세포 - 세포 상호 작용의 조사를하는 데 도움이 될 수 있습니다.

Watch this Scientific Journal Video about Using Laser Tweezers For Manipulating Isolated Neurons In Vitro at JoVE.com

Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

이 동영상은 체외에서 레이저 핀셋을 사용하여 교양 뉴런의 조작을 설명합니다.

체외에서 분리된 뉴런을 조작에 대한 레이저 족집게를 사용하여

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult ...

using-laser-tweezers-for-manipulating-isolated-neurons-in-vitro

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.3K 조회수.  University of Medicine and Dentistry of New Jersey - UMDNJ. 이 동영상은 체외에서 레이저 핀셋을 사용하여 교양 뉴런의 조작을 설명합니다.

비디오: Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

FM 레이블을 사용하여 뉴런의 Exocytosis 측정

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

연구

생물학

Silicon Microchips for Manipulating Cell-cell Interaction

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from development to malignant transformation.&nbsp; In particular, interaction with neighboring stroma has been implicated in a plethora of biological phenomena; however, conventional techniques limit the ability to interrogate the spatial and dynamic elements of such interactions.
In Micromechanical Reconfigurable Culture (RC), we employ a micromachined silicon substrate with moving parts to dynamically control cell-cell interactions through mechanical repositioning. Previously, this method has been applied to investigate intercellular communication in co-cultures of hepatocytes and non-parenchymal cells, demonstrating time-dependent interactions and a limited range for soluble signaling 1.
Here, we describe in detail the preparation and use of the RC system. We begin by demonstrating the handling of the device parts using tweezers, including actuating between the gap and contact configurations (cell populations separated by a narrow 80-&micro;m gap, or in direct intimate contact). Next, we detail the process of preparing the substrates for culture, and the multi-step cell seeding process required for obtaining confluent cell monolayers. Using live microscopy, we then illustrate real-time manipulation of cells between the different possible experimental configurations. Finally, we demonstrate the steps required in order to regenerate the device surface for reuse: toluene and piranha cleaning, polystyrene coating, and oxygen plasma treatment.

This article describes an experimental approach for dynamic regulation of cell-cell interactions between adherent cells on a micrometer scale. Manipulation of intercellular communication between hepatocytes and stromal cell is demonstrated. The developed platform enables investigation of cell-cell interactions in a variety of biological processes, including development and pathogenesis.

세포 세포 상호 작용을 조작에 대한 실리콘 마이크로 칩

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from ...

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

진단 도구를 개발하기 위해 마이크로 전자 기계 시스템 (MEMS)를 사용

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

사용 Schwann - astrocyte 상호 작용 분석 체외에서 Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Ribosome 바운드 초기 Polypeptides의 분리<em&gt; 체외에서</em&gt; mRNA 함께 Translational 일시 중지 사이트를 식별하려면

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

생체 외 분석에서 집계 Hyperphosphorylated 타우 단백질을 사용하여

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Manipulating the Murine Lacrimal Gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.

The lacrimal gland (LG) is a branching organ that produces the aqueous components of tears necessary for maintaining vision and ocular health. Here we describe murine LG dissection and ex vivo culture techniques to decipher signaling pathways involved in LG development.

쥐의 눈물샘을 조작

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

마우스입니다 관류 신장 기법

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

스펙트럼 유동 세포 계측법에 의해 고체 조직에서 분리 한 세포 현탁액의 분석

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

체외에서 분리된 뉴런을 조작에 대한 레이저 족집게를 사용하여

요약

더 많은 비디오 탐색

이 비디오의 챕터