Araştırma NIH Grants EY012031 ve EY0182175 ve FM Kirby Vakfı tarafından desteklenmiştir.

Izole kültür hücreleri işlemek için Optik cımbız Optik cımbız Yakalama güçleri ışık ivme (Ashkin ve ark, 1986 Ashkin, 1991) oluşturulur. Bu güçlerin kolayca süspansiyon hücreleri tuzak olsa da, onlar bir yüzeye bağlı hücreleri hareket etmek mümkün değildir. Bu nedenle, hücrelerin tuzağa noktada kültür çanak yapışma azaltmak için, lamel itici hücre ile poli-2-hydroxyethylmethacrylate (poli-HEMA) (Sigma Chemical Co, St Louis, MO), toksik olmayan bileşik kaplı özellikleri daha önce endotel hücreleri (Folkman ve Moscona, 1978) yapışma çalışmaları için çalışmaktadır. Poli-HEMA bir kültür yemeğin yarısını kapsar, diğer yarısı hücre büyümesini destekleyen bir substrat ile kaplanmıştır. Laboratuvarımızda, Sal-1, kültür semender retina hücreleri için substrat olarak kullanılır . Sal-1 imalatı ve poli-HEMA kaplı yemekleri aşağıda açıklanan prosedürü: Kültür yemekleri imalatı <ol><li> Temizlik coverglasses asit (# 1 VWR Bilimsel A.Ş., Medya, PA). Bu coverglass lazer odaklanma için gerekli yüksek sayısal açıklık hedefleri için gerekli olan sadece 0.1mm kalınlığı için seçildi. </li><li> Bir hücre itici alanı oluşturmak için, oda sıcaklığında bir gecede% 95 etanol 1 ml 20 mg poli-HEMA (Sigma Chemical Co, St Louis, MO) çözülür. Tercihen, bu çözüm hazırlık 1 ay içinde kullanılması gerekir. </li><li> Yakın dikey konumda bir başlık gibi tozsuz bir ortamda coverglasses Prop. </li><li> 200μl mikropipet plastik ucundan coverglass en yakın poli-HEMA çözümün bir veya iki damla yerleştirin. Damla (her 50-100μl hakkında) coverglass bir yarısında sınırlı olmalıdır. Eğimli sıvı ince bir poli-HEMA bile kaplama, alt coverglass yüzeyi aşağı akar sağlar. Poli-HEMA kaplı coverglasses kaput oda sıcaklığında 30-60 dakika kurumasını bekleyin. </li><li> Coverglass poli-HEMA yan kenarına yakın bir ince uçlu kalem ile işaretleyin. </li><li> 35 mm kültür çanak alt kısmında 1 cm delik. </li><li> Tutkal, poli-HEMA kaplı yan yemeğin içine dönüktür Sylgard 184 (Dow Corning Co, Midland) ile kültür çanak alt poli-HEMA kaplı coverglass. Poli-HEMA kaplama kenar olun deliğin merkezi çalışır. Yemekleri, tozsuz bir ortamda oda sıcaklığında 1-3 gün boyunca kurumasını bekleyin. </li><li> Poli-HEMA kaplama kenarında bir elmas uçlu kalem ile coverglass dışında bir çizgi kazıyıp. Ardından dışında yaklaşık 2mm dik açıda iki geçiş çizgileri çizebilir. Iki kesişme noktası, referans mikroskop sahnede kültür çanağı aynı yönde yeniden kullanılmak üzere fiducial noktaları olarak hizmet vermektedir. </li><li> Bulaşıkları gecede ultraviyole ışık altında sterilize edin. </li><li> , Retinal hücre zarlarının (cömertçe Dr. Peter MacLeish Morehouse School of Medicine tarafından sağlanan karşı yükseltilmiş bir fare anti-semender antikor içeren bir hücre kültürü çanak-yapışık yarısı olmayan Sal-1 ile poli-HEMA yarım kat, oluşturmak için Atlanta, GA. MacLeish ve ark (1983) bakınız). İlk olarak, ceket, yaklaşık 100μl 0.1 mg / ml steril keçi anti-fare IgG (Corporation&#39;ın Boehringer Mannheim, Indianapolis IN) Sal-1 tarafı, poli-HEMA yüzüne üzerine sıvı dökmemeye özen. Üç saat bekletin, sonra çıkarın. Bir veya iki kez, daha sonra çanağı poli-HEMA yan içine sıvı dökmemeye özen 100μl Sal-1 süpernatant yerde aynı alanda steril Ringer solüsyonu ile yıkayınız. Sal-1, bilinen bir konsantrasyonda kaplı ilk antikor sağlar. </li><li> Sal-1 gecede yemekler inkübe edin. </li><li> Sal-1 antikoru çözüm çıkarın ve steril Ringer solüsyonu ile bölgeyi durulayınız. Hücre kaplama hemen önce yemek için 2 ml orta tanıtın. </li></ol> Amfibi Ringer solüsyonu, serum serbest amfibi, orta ve enzim sindirim için Ringer solüsyonu kompozisyon MacLeish ve Townes-Anderson (1988) ve Mandell ve ark. (1993). Hücre kültürlerinin hazırlanması Bu video kullanılan hücreler elde edilmiştir ışık adapte, yetişkin, su fazlı kaplan semenderlerin (Ambystoma tigrinum), uzunluğu 17-22 cm (Charles Sullivan Inc, Nashville, TN), 5 muhafaza ° C 12 saat: 12 saat ışık-karanlık döngüsü. Protokolleri Kurumsal Hayvan Bakım tarafından onaylanan ve Ulusal Sağlık Enstitüsü&#39;nün kurallarına sıkı sıkıya bağlı bir New Jersey Tıp ve Diş Hekimliği Üniversitesi Komitesi (IACUC) kullanın. Retinal hücre kültürlerinin hazırlanması için prosedürler Nachman-Clewner ve Townes-Anderson (1996) tarafından açıklanan ve aşağıdaki gibidir: <ol><li> Hayvanların başını kesmek ve ilik. Gözlerinizi çıkarın ve kornealar ve iris teşrih. Retina Göz yuvası içinde biraz ayırmak gerekir. Kavisli bir forseps retina altına yerleştirerek, retina kepçe. Lens bu aşamada retina bağlıysa önemli değildir. </li><li> 40 dakika için 14 U / ml semender Ringer solüsyonu papain (Worthington, Freehold, NJ) retina Digest. </li><li> Kullanmadan önce, papain Ringer solüsyonu 30 dakika boyunca% 95 O 2 /% 5 CO 2 gaz ile kabarmış olmalı, daha sonra 0.22 mikron filtre (Millipore, Carrigtwohill, İrlanda) geçişi ile sterilize . </li><li> Steril semender Ringer solüsyonu üç kez retina durulayın. </li><li> Ringer çözüm lensleri çıkarın, sonra yavaşça bir hücre süspansiyonu verim pipet delik 3 mm ile retina dokusunun çiğnemek. </li></ol> Lazer cımbız manipülasyon izole nöronların <ol><li> , Tabak sahnede bir referans noktası ile bir işareti hizalayarak çanak yönlendirilmesi, mikroskop sahnede kültür tabağına yerleştirin. Koordinatları bilgisayara fiducial puan kaydedin. </li><li> Plaka Sal-1 hem de üzerine hücreleri ve çanak poli-HEMA yarısı. </li><li> Hücrelerin antikor substrat eklemek için çanak yapışık tarafta hücreler için yeterince uzun olan, yaklaşık 20 dakika boyunca yerleşmek için izin verin. </li><li> Sal-1 yapışık yemeğin tarafında bir hücre seçin. X ve y aşamada hücre yakın bir konumda koordinatları işaretleyin. Bizim durumumuzda, nokta seçili hücre birincil dendrit yaklaşık 10μm. Koordinatları bilgisayara kaydedin. </li><li> Yemeğin poli-HEMA tarafında bizim durumumuzda bir fotoreseptör ikinci bir nöron, seçin ve onu yakalamak için lazer cımbız aracını kullanın. Yakalama, geniş bir aralıkta güç seviyeleri elde edilebilir. Ancak, biz% 10 hücre ile birlikte yakalama enkaz önlemek için yeterince düşük -20%, lazer güç, ancak orta yoluyla hücre taşınması için yeterli. </li><li> Tuzak hücre tutarken, böylece hücre kültür çanak yüzeyinden ve herhangi bir bağlı nöronlar üzerinde evresini. Sal-1 tarafında önceden kaydedilmiş x ve y koordinatları hücre getirmek için bilgisayar kontrolü altında sahne taşıyın. Sahne hareketi 8-20μm / s olarak belirlendi, ancak maksimum hız ampirik olarak tespit edilmelidir. Sal-1 tarafındaki varış noktasına vardıklarında, çanak yüzeyinde sıkışıp hücre getirmek için sahne yükseltmek. Hücre yerleştirme birkaç mikron hassasiyetle yapılabilir. Sal-1 substrat hücre uymak için izin ver. </li><li> Çifti oluşumu öncesi ve sonrasında çiftin iki hücrelerinin sayısallaştırılmış görüntü elde edilmesi yararlı bir kayıt sağlar. </li><li> Bir kuluçka hücre çiftleri koruyun. Semender hücreleri için, 10 nemlendirilmiş bir odasında yemekleri ° C Hücreler video zaman atlamalı tarafından izlenmekte ve gerekli her türlü hücre biyolojik tekniği ile kullanılabilir. </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>

Işık momentumu vardır ve bir hücre geçerken kırılan bir ışık ışını zaman, bir kuvvet momentum yönünü değiştirmek için gerekli. Çünkü momentumun korunumu yasası, ters yönde bir kuvvet, sırayla, bir hücre tepki vermelidir. Ashkin (1991) mikroskop objektif lens ile odaklanmış bir lazer ışını tarafından üretilen kuvvet, bir hücre odak merkezine doğru hareket edeceğini gösterdi. Bir lazer ışını sadece birkaç piconewtons güç üretir rağmen, bu gücün, orta aracılığıyla bir hücre çekmek için y...

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		<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

Bu kağıt ve video, hedefleme tercihleri, in vitro yetişkin retinal nöronların yenileyici hücre süreçlerini incelemek için laboratuarda kullanılan protokoller açıklanmaktadır. Kaplama ile izole retina hücrelerinin özellikle lazer cımbız ile kullanımı için yapılan yemekler diseksiyonu, sindirim ve retina, öğütme ve bitiş retinal hücre kültürlerinin hazırlanması için prosedürler ile başlar. Bu yemekleri bir hücre yapışkan yarım ve bir hücre itici yarısı ayrılır. Hücre yapıştırıcı yan hücrelerin büyümesine üzerine bir substrat sağlarlar Sal-1 antikorları, bir tabaka ile kaplanmıştır. Diğer yapışkan substratlar diğer hücre türleri için kullanılabilir. Hücre itici tarafı poli-HEMA ince bir tabaka ile kaplıdır. Yemeğin poli-HEMA tarafında kaplama hücreleri, lazer cımbız ile tuzağa taşınan ve ardından bir çift oluşturmak için bir hücre Sal-1 tarafta komşu olarak yerleştirilmelidir. Bu teknik ile herhangi bir boyut hücre gruplarının oluşumu mümkün olmalıdır. &quot;Lazer cımbız kontrollü mikromanipülasyon&quot; araştırmacı hücreleri taşımak için hangi seçim anlamına gelir, ve hücreler arasındaki istenen mesafe standart olabilir. Çünkü lazer ışını kültür çanağı şeffaf yüzeylerden geçer, hücre seçme ve yerleştirme, kapalı, steril bir ortamda yapılır. Hücreler video time-lapse tarafından izlenmekte ve gerekli her türlü hücre biyolojik tekniği ile kullanılabilir. Bu teknik, hücre-hücre etkileşimleri soruşturma yardımcı olabilir.

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

Bu video in vitro lazer cımbız kullanarak manipülasyon kültürlü nöronların açıklamaktadır.

In Vitro İzole Nöronlar kurgulama için Lazer Cımbız

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

Research

JoVE Journal

Biology

10.3K Görüntüleme.  University of Medicine and Dentistry of New Jersey - UMDNJ. Bu video in vitro lazer cımbız kullanarak manipülasyon kültürlü nöronların açıklamaktadır.

Video: 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.

Nöronlar FM Etiketleme kullanarak ölçüm ekzositoz

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 ...

Biyoloji

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.

Hücre hücre Etkileşim kurgulama için Silikon mikroçipler

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

Teşhis Araçları Geliştirmek İçin Mikro-Elektro-Mekanik Sistemler (MEMS)

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.

Schwann-astrosit Etkileşimleri Analizi In Vitro Tahliller

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.

Ribozom Bağlı Doğan Polipeptitler İzolasyonu<em&gt; In vitro</em&gt; MRNA boyunca Translasyonel Duraklat Siteleri Belirleme

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.

In Vitro toplama Deneyler hiperfosforile tau proteini kullanılarak

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.

Kemirgen Gözyaşı bezi kurgulama

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.

Fare İzole perfüze Böbrek Tekniği

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.

Spektral Sitometrisi ile Katı Dokular İzole Hücre Cezalar Analizi

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.