This work was supported by National Eye Institute Grant R01 EY017724 (VMF) and National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant K99 AR066534 (DSG).

Bütün hayvan prosedürleri Scripps Araştırma Enstitüsü&#39;nde Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından Sağlık ve onaylı protokol çerçevesinde Ulusal Sağlık Enstitüleri tarafından Laboratuvar Hayvanları Bakım ve Kullanımı için Kılavuzu&#39;nda tavsiyeleri doğrultusunda yapıldı. 1. Mercek Diseksiyon <ol><li> Sağlık &quot;Laboratuvar Hayvanları Bakım ve Kullanım Kılavuzu&quot; National Institutes ve onaylanmış kuruluş hayvan kullanım protokolleri önerilere göre fareler Euthanize. </li><li> Kavisli bir forseps kullanılarak farelerden göz aydınlatmak. soketten göz getirmek ve daha sonra forseps ile soketten göz koparmak için forseps ile göz etrafında doku basınız. diseksiyon çanak taze 1x fosfat tamponlu salin (PBS) gözleri aktarın. </li><li> mümkün olduğunca göz küresinin kadar yakın optik siniri kesti. Yavaşça ve dikkatlice delik nereye yoluyla küresi içine ince düz bir cımbız eklemeke optik sinir posterior çıkar. </li><li> Dikkatle korneanın kenarından posterior göz küresi içine makas ile bir kesi yapmak. Kemirgen lensler gözün ~% 30 işgal. dikkatle bu kesiler yapmak ve lensi zarar vermemek için çok derin göz içine cımbız veya makas takmayın. </li><li> göz küresi etrafında en az yarım kornea ve sklera arasındaki kavşakta boyunca kesilir. </li><li> Yavaşça adımlarda 1.4 ve 1.5 yapılan açıklıktan göz merceği kaldırmak için kornea üzerinde itin. </li><li> dikkatle hâlâ lense bağlı herhangi bir büyük enkaz kaldırmak için ince uçlu düz forseps kullanın. Görme sertlik ölçümlerine geçmeden önce herhangi bir hasar lensi kontrol edin. </li></ol> 2. Sertlik Ölçümleri <ol><li> bir analitik terazi kullanarak aynı kutudan en az 10 lamelleri tartılır. lamelleri ortalama ağırlığını bulun. Tutarlılık için, tüm deneyler için lamelleri aynı kutusunu kullanın. Ön-yaşDeney başlamadan önce en az 2 saat oda sıcaklığında 1 x PBS içinde lamelleri ve sağ-açılı ayna. </li><li> 1x PBS 75 ml - ölçüm gözüne doldurun 65 ile (Şekil 1 e bakınız). Ölçüm odası bir in-house makine atölyesi tarafından pleksiglas üzerinden yapıldığı ve odasında Divots uygun bir matkap ucu ile istenilen derinliğe kadar ayarlanmış bir matkap basın tarafından yapılmıştır. Lensler mekanik test süresince, oda sıcaklığında 1 x PBS içinde saydam kalır. </li></ol><img alt="Şekil 1" src="/files/ftp_upload/53986/53986fig1.jpg" /> Şekil 1:. Sertlik Ölçüm Odası farklı derinliklerde ve şekiller divots çeşitli ısmarlama sertlik ölçüm odasının boyutlarını gösteren bir fotoğraf. 200 mikron veya 300 mikron derin (sarı ok başları) olan yuvarlak Divots fare lensler üzerinde ölçümler için kullanılmaktadır. Divots 2 mm çapında ve ~ 13 vardır-. Odanın kenarından 14 mm <a href="https://www.jove.com/files/ftp_upload/53986/53986fig1large.jpg" target="_blank">bu rakamın daha büyük bir versiyonunu görmek için lütfen buraya tıklayınız.</a> <ol start="3"><li> lens tutmak için kullanılacak çimen sabit bir mesafede odasına dik açılı ayna yerleştirin. Ayna deney sırasında hareket olmadığından emin olun. </li><li> dikkatle ele geçirmek forseps veya kavisli bir forseps ile ölçüm odasına transfer disseke lensler. </li><li> tepeme gelen yüksüz lens bir üst görünüm resim çekin. mikroskop (altta) ve sol ve sağ tarafta bir fiber optik ışık kaynağı diseksiyon gelen aydınlatma ile 30X büyütmede fare lens fotoğraf çekmek. Maksimum ışık yoğunluğu% 80 fiber optik güç kaynağı olarak ayarlayın. gerektiği gibi ortam aydınlatması, kullanıcı tercihi ve resim kalitesine dayalı güç kaynağı çıkışını ayarlayın. </li><li> ri aracılığıyla görülebilir yüksüz lensin yan görünüm resim çekinGHT-açılı ayna. Kamera kalibre değilse, odak ayna kenarına bir resim çekmek. Ayna kenarı 5 mm uzunluğunda ve bu ölçü daha sonra piksel / mm belirlemek ve görüntülerde bir ölçek çubuğu olarak hizmet için kullanılabilir. </li><li> Yeri çimen içine lens ve lens çimen güvenli ve düz oturduğundan emin olun. yüklemeden önce lensin bir fotoğrafını çekin. lens, ön veya arka direğe çimen istirahat edilmelidir. </li><li> lens üzerine yavaşça 1 lamel yerleştirin. sürünme izin vermek için 2 dakika bekleyin ve yüklü lens başka yan görünüm resim çekmek. </li><li> Adım 2.8 gibi lamelleri ekleme ve 10 lamelleri toplam uygulanır kadar adım 2.8 gibi her lamel eklenmesinden sonra yan görünüm Fotoğraf çekmeye devam edin. </li><li> Tüm lamelleri çıkarın. 2 dakika bekleyin ve tüm lamelleri çıkardıktan sonra lens (iç ve dış çimen ve) bir yan bakışı resim çekmek. </li></ol> 3. Objektif Nucleus Ölçümü <ol><li> unsurlar içinLens çekirdeği boyutu ine, 1x PBS ile dolu temiz bir Petri kabı objektif hareket. </li><li> Yavaşça ince düz forseps kullanarak lensi açarlar. </li><li> Slough off eldivenli parmakları arasında merceği yuvarlayarak kortikal lif hücreleri. Kalan lens çekirdeği sert bir mermer gibi hissedeceksiniz. yaş 1 ay başlayan yetişkin lensler üzerinde çekirdeğini izole etmek için bu yordamı kullanın. İzole çekirdeği katı bir cisim olduğundan, mercek çekirdeğin ileri mekanik test, bu anlatılan yöntemi kullanarak yapılamaz. </li><li> Yavaşça Petri kabındaki 1x PBS lens çekirdeği durulayın. </li><li> geri ölçüm odasına (değil çimen olarak) içine lens çekirdeği koyun ve dik açılı ayna aracılığıyla mercek çekirdeğinin bir görüntü alır. </li></ol><img alt="şekil 2" src="/files/ftp_upload/53986/53986fig2.jpg" /> Şekil 2:. Ex lameller tarafından Sıkıştırılmış bir fare Lensler (A) şematik ve (B) fotoğraf1x PBS ile dolu ölçüm odasında 200 mikron derinliğinde çimen içinde 2 aylık fare lensi gösteren deneysel bir kurulum. Bir dik açı ayna ve bir diseksiyon mikroskobu üzerine monte edilmiş bir dijital kamera lamelleri ile sıkıştırma sırasında lens görüntüleri toplamak için kullanılmıştır. (C) arka arkaya lamelleri artan sayıda sıkıştırılan 2 aylık vahşi tip lensin sagital görüş Fotoğraflar eksenel ve ekvatoral çapları ölçülerek ve lamel tabanlı sıkıştırma testi sırasında eksenel ve ekvatoral suşları hesaplamak için ham veri sağladı. lensin bir yansıması zaman lamelleri (çoğu açıkça görülebilir 1 lamel görüntüde) görülebilir. ölçümleri yaparken, yansıma görmezden ve lensin apekse ölçün. (D) 2 aylık vahşi tip objektif sonrası sıkıştırma ve izole objektif çekirdeğin sagital manzarası fotoğrafları. Post-sıkıştırma objektif ve izole çekirdek çimen dışında oturuyor. Ölçek çubukları, 1mM. Bu rakam, gökh modifiye edilirve ark. PLoS biri 2012 yılında 19. <a href="https://www.jove.com/files/ftp_upload/53986/53986fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> 4. Görüntü Analizi <ol><li> yüklemeden önce ve ImageJ veya benzer bir yazılım kullanarak her yükleme aşamasından sonra lenslerin ekvatoral ve eksenel çaplarını ölçün. Her mercek çekirdeğinin çapını ölçün. Herhangi bir yönde bir ölçüm 19,21 yeterli olacaktır bu yüzden objektif çekirdeği neredeyse küreseldir. </li><li> kullanılan çimen derinliğini ekleyerek çapları eksenel lensi düzeltin. Ölçüm odasında, çimen lens eksenel kalınlığı 200 mikron (2 aylık fare lensler) ya da 300 mikron (4 aylık ve 8 aylık fare lensler) gizlenmiş. </li><li> , Ε = (d - d 0) denklem kullanılarak lens çapı ölçümleri eksenel ve ekvatoral suşları hesaplayın ε suşu / d, 0, d eksenel veya equatorial belirli bir yük altında çapı ve d, 0, sıfır yükte karşılık gelen eksenel veya ekvatoral çapıdır. </li><li> (Mg olarak) uygulanan yükün fonksiyonu olarak eksenel ve ekvatoral suşları çizilir. </li><li> eksenel Ekvator ve nükleer çapları çiziniz. Hesaplayın ve ekvator çapı eksenel çapı bölerek mercek boy oranını arsa. </li><li> Hesaplamak ve denklem kullanılarak objektif hacmini arsa hacmi = r E Ekvator yarıçapı ve r A adımında 2.6 alınan resimden ölçülen eksenel yarıçap olduğunu 4/3 × π × r E 2 × r A,. Bu denklem objektif bir basık küremsi (elipsoid) 1,22 olduğunu varsayar. </li><li> Hesaplayın ve adım 3.5 alınan resimden ölçülen r N objektif çekirdeğinin yarıçapı r N 3, × denklemi, hacim = 4/3 × π kullanarak nükleer hacim arsa. Bu denklem lens çekirdeği i varsayarsa küre 19,21. </li><li> Hesaplayın ve lens hacmine nükleer hacim oranı olarak nükleer fraksiyonu arsa. </li></ol>

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermediate+filaments+regulate+tissue+size+and+stiffness+in+the+murine+lens.">Intermediate filaments regulate tissue size and stiffness in the murine lens.</a> Invest Ophthalmol Vis Sci. 52 (6), 3860-3867 (2011).</li><li>Kuszak, J. R., Mazurkiewicz, M., Zoltoski, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+modeling+of+secondary+fiber+development+and+growth:+I.+Nonprimate+lenses.">Computer modeling of secondary fiber development and growth: I. Nonprimate lenses.</a> Mol Vis. 12, 251-270 (2006).</li><li>Scarcelli, G., Kim, P., Yun, S. 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Yazarlar ifşa hiçbir şey yok.

Lens sertliği ölçmek için bu yöntemi kullanırken birkaç önemli hususlar vardır. odanın (θ) alt kısmına göre - (8.5 ° 8) İlk olarak, lamelleri hafif eğik bir açıyla lens uygulanır. Bu da, eksenel fazla ekvatoryal yerine yük çok küçük bir bileşen uygulanır. Ancak, bu ekvatoral yük günah θ ≈ 0.1 19 çünkü önemsiz kabul edilir. Bu yöntem büyük lensler için adapte edilirse, odasının tabanına lamelleri açısı ekvator yük gerilme hesaplamaları hesaba gerekip gerekmediği...

The lens is a transparent and avascular organ in the anterior chamber of the eye that is responsible for fine focusing of light onto the retina. A clear basement membrane, called the lens capsule, surrounds a bulk of elongated fiber cells covered by an anterior monolayer of epithelial cells1,2. Life-long growth of the lens depends on the continuous proliferation and differentiation of epithelial cells at the lens equator into new fiber cells that are added onto previous generations of fiber cells in a concentric manner2. Over time, lens fiber cells undergo compaction, resulting in a rigid center in the middle of the lens called the nucleus1. Accommodation, defined as a dioptric change in the optical power of the eye, occurs in humans when the ciliary muscles contract to deform the lens and allow a true increase in optical power to focus on near objects3-5. In the unaccommodated eye, the lens is held in a relatively flattened state due to tension from zonular fibers. When the ciliary muscles contract, the tension on the lens is released, leading to decreased lens equatorial diameter and increased axial thickness. Age-related changes in the lens cause presbyopia, a progressive loss of lens accommodation, which leads to the need for reading glasses. 
Several studies have linked presbyopia to age-related increase in the intrinsic stiffness of the lens6-11. Stiffness is defined as the resistance of an elastic object to deform under applied load. A variety of methods have been used to examine stiffness of human lenses, including spin compression12-14, actuator compression15, probe indentation16, dynamic mechanical analysis 6,10 and bubble-based acoustic radiation force17. While mouse lenses do not accommodate or develop presbyopia, mouse models for lens pathologies are valuable tools because mice are less expensive than larger animals, well characterized genetically and undergo accelerated age-related changes due to rapid aging. A handful of studies have examined mouse lens stiffness with compression methods and demonstrated changes in lens stiffness due to aging or targeted genetic disruptions18-21. Thus, mouse lenses are good models for studying age-related changes in lens stiffness. 
This protocol describes a simple and inexpensive, yet precise and reproducible, compression method for determining mouse lens stiffness based on application of glass coverslips onto the lens in conjunction with photographing the lens through a dissection microscope and mirror. This method yields robust strain and morphometric data with an easily fabricated and assembled apparatus. The representative results confirm that mouse lenses increase in stiffness with age.

<table border="0" cellpadding="0" cellspacing="0" width="811">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Fine tip straight forceps</td>
			<td style="width:139px;">Fine Scientific Tools</td>
			<td style="width:119px;">11252-40</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microdissection scissors, straight edge</td>
			<td style="width:139px;">Fine Scientific Tools</td>
			<td style="width:119px;">15000-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved forceps</td>
			<td style="width:139px;">Fine Scientific Tools</td>
			<td style="width:119px;">11272-40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Seizing forceps</td>
			<td style="width:139px;">Hammacher</td>
			<td style="width:119px;">HSC 702-93</td>
			<td>Optional</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection dish</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">12565154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60mm petri dish</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">0875713A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X phosphate buffered saline (PBS)</td>
			<td style="width:139px;">Life Technologies</td>
			<td style="width:119px;">14190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18mm x 18mm glass coverslips</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">12-542A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Measurement chamber with divots to hold lenses</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Custom-made (see Figure 1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Right-angle mirror</td>
			<td style="width:139px;">Edmund Optics</td>
			<td style="width:119px;">45-591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light source</td>
			<td style="width:139px;">Schott/Fostec</td>
			<td style="width:119px;">8375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Illuminated dissecting microscope</td>
			<td style="width:139px;">Olympus</td>
			<td style="width:119px;">SZX-ILLD100</td>
			<td>With SZ-PT phototube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital camera</td>
			<td style="width:139px;">Nikon</td>
			<td style="width:119px;">Coolpix 990</td>
			<td></td>
		</tr>
	</tbody>
</table>

sequential application of glass coverslips to assess the compressive stiffness of the mouse lens: strain and morphometric analyses

Göz merceği kırar ve retinada net bir görüntü oluşturmak için ışık odaklanan şeffaf bir organdır. İnsanlarda, siliyer kaslar kasılır yakındaki nesnelere odaklanmak lens optik güç, konaklama olarak bilinen bir süreç içinde bir artışa yol açarak, lensi deforme etmek. Lens sertliği yaşa bağlı değişiklikler presbiyopi bağlantılı olmuştur, uzantısına göre karşılamak için objektif yeteneğini azalma ve, okuma gözlükleri için gereklidir. Fare lensler karşılamak ya da presbiyop geliştirmek değil olsa bile, fare modelleri anlayış lens patolojileri için paha biçilmez bir genetik araç sağlayabilir ve farelerde gözlenen hızlandırılmış yaşlanma lens yaşa bağlı değişikliklerin çalışmayı sağlar. Bu protokol, fare lens sertliği belirleyen sırayla uygulamak lens üzerine sıkıştırma yüklerini artan cam lamelleri kullanarak, basit, hassas ve düşük maliyetli yöntem gösterilmektedir. Temsilcisi veri fare lensler gibi, yaşla birlikte daha sert hale teyitİnsan lensler. Bu yöntem son derece tekrarlanabilir ve potansiyel büyük hayvanlar test lensleri mekanik olarak kadar ölçeklendirilebilir.

sertliği ve 2, 4 ve 8 aylık bir fare lenslerin boyutlarının ölçüldü. Fareler TSRI Hayvancılık Tesisi elde edilen saf C57BL6 suşu arka planda tüm yabani tip hayvanlar vardı ve her lens 1 ila 10 lamelleri ile yüklendi. eksenel ve ekvatoral suşları her lamel ilave edildikten sonra lensin eksenel ve ekvatoral çapları ölçülerek ve daha sonra karşılık gelen boş çapa çapında her değişikliği normalize ederek uygulanan yükün bir fonksiyonu olarak hesaplandı. Her ...

Watch this Scientific Journal Video about Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses at JoVE.com

Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses

Fare Lens Basınç Sertliği değerlendirin Cam lameller Sıralı Uygulama: Gerilme ve Morfometrik Analizleri

Age-related increases in eye lens stiffness are linked to presbyopia. This protocol describes a simple, cost-effective method for measuring mouse lens stiffness. Mouse lenses, like human lenses, become stiffer with age. This method is precise and can be adapted for lenses from larger animals.

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the ...

sequential-application-glass-coverslips-to-assess-compressive

Research

JoVE Journal

Biology

7.2K Görüntüleme.  The Scripps Research Institute. Age-related increases in eye lens stiffness are linked to presbyopia. This protocol describes a simple, cost-effective method for measuring mouse lens stiffness. Mouse lenses, like human lenses, become stiffer with age. This method is precise and can be adapted for lenses from larger animals.

Video: Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

Zebra balığı Lens Nakli ve Göz Mutants Analizi Uygulaması

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Biyoloji

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Saflaştırılması M. magneticum Deformasyon AMB-1 Magnetosome İlişkili Protein MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

Proteomik Analizleri Fare Vitreus ve Retina eviserasyon

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

Fazlararası Hücre Çekirdek Mekanik Özellikleri Probe Biyofiziksel Tahliller: Yüzey Gerilme Uygulama ve Microneedle Manipülasyon

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

İsteyerek Pluripotent Kök Hücrelerinin Elde Edilen Fare Üretimi

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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

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

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

Gap Junctional Hücreler arası iletişim: Doğal Ürünler Toksikantların ve Toksinler Yan Etkileri değerlendirmek için Fonksiyonel Biyomarker ve Sağlık Faydaları

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

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.

sertliği ölçme<em&gt; Ex Vivo</emAtomik Kuvvet Mikroskobu Kullanma&gt; Fare aorta

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

A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the structural and cellular components of the mandibular condylar cartilage (MCC). This manuscript describes methods for analyzing these changes and a method for altering the loading of the TMJ in mice (i.e., compressive static TMJ loading). The structural evaluation illustrated here is a simple morphometric approach that uses the Digimizer software and is performed in radiographs of small bones. In addition, the analysis of cellular changes leading to alterations in collagen expression, bone remodeling, cell division, and proteoglycan distribution in the MCC is described. The quantification of these changes in histological sections - by counting the positive fluorescent pixels using image software and measuring the distance mapping and stained area with Digimizer - is also demonstrated. The methods shown here are not limited to the murine TMJ, but could be used on additional bones of small experimental animals and in other regions of endochondral ossification.

This manuscript presents methods for analyzing morphometric and cellular changes within the mandibular condyle of rodents.

Bir xarakteristikaları ve fare çene kemiğinde düzgün için hücresel analiz yöntemi

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

Dinamik için objektif-ücretsiz Video mikroskobu ve kantitatif analiz yapisan hücre kültürü

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

Elektrofizyoloji ölçüm uygulanması elektro-tarafsız taşıyıcılar etkinlik çalışması için

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...