Teitell laboratuar İnsan embriyonik kök hücre çalışmaları California Rejeneratif Tıp Enstitüsü (CIRM) tohum hibe RS1-00313 tarafından desteklenmektedir. Geniş, UCLA, özellikle Dr. Amander Clark, Dr Jerome Zack ve verdikleri destek için çalışmalarımız UCLA Broad Enstitüsü Kök Hücre Çekirdek Tesis üyeleri Rejeneratif Tıp ve Kök Hücre Araştırma Merkezi üyelerine teşekkür ederiz.

Matrigel Matrigel için Yarma hESC Genellikle Matrigel üzerinde hESC bir konfluent 6 plaka, kuyu, yarma, 4-5 gün sonra tekrar konfluent olma, başka bir Matrigel plaka 1:5 için 1:3 bölünmüş olabilir. <ol><li> Yarma önce yukarıda açıklandığı gibi, CM ve Matrigel plakaları hazırlanır. </li><li> Bölme gününde, × 1 ile bölme PBS, pH7.4 her iyi yıkayın iyi 1mg/ml Dispase (DMEM/F12 medya ile seyreltilmiş 5mg/ml stok solüsyonu) 1 ml ve 37 inkübe ° C 3 dk. </li><li> 1 ile her iyi üç kez yıkayın × PBS, pH7.4,, ES medya, 1ml bFGF olmadan eklemek iyi alt kapalı koloniler kazımak için bir hücre kazıyıcı, 50ml şahin tüp askıya hESC toplamak ve santrifüj yoluyla pelet 200g oda sıcaklığında 5 dakika. </li><li> Matrigel plakalar üzerinde yeniden plakalı hESC, ilk DMEM/F12 ile Matrigel tabak yıkama ve başına 10 ng bFGF ile desteklenmiş CM 2.5ml ekleyin. 10 ng bFGF, pipet hücre süspansiyonu yukarı ve aşağı üç kez ile desteklenmiş CM uygun bir hacmi hESC pelletini tekrar. (NOT: yarma için hESC Matrigel plakaları geldiğinde, yığınları kırmak için çok kolay, bu nedenle üç tur pipetleme yeterli ve daha koloniler üzerinde bozabilir ve tek hücre yapabilir pipetleme.) Temsili miktar 0.5 ml ortalama hESC kümeleri süspansiyon 6-iyi Matrigel plaka nihai bir birim olarak ortalama 3ml ulaşmak için. 37 ° C inkübatör plaka koyun. </li></ol> Immünofloresan mikroskopi pluripotency belirteçlerin saptanması İmmünofloresan mikroskopi, kültür sırasında hESC pluripotency belirteçlerinin Ekim-4 ve SSEA-4 ifadesi incelemek için kullanılır. <ol><li> Iyi bir medya aspire ve 1 ile yıkayın × PBS, pH 7.4. 10 dakika düzeltmek için iyi,% 3.7 formaldehit 2ml ekle. </li><li> 2 dakika süreyle iyi yer ve -20 ° C 2 ml metanol eklemek, 1xPBS pH 7.4 ile durulayın. </li><li> 5 dakika süreyle% 0.2 sığır serume albumin (BSA) 1ml 1xPBS, pH 7.4 ve blok ile durulayın. </li><li> Ekim-4 veya anti-h/mSSEA-4 antikor (% 0,2 BSA 1:200 dilüsyon) 1 ml ekleyin ve 1 saat veya 4 ° C&#39;de gece boyunca oda sıcaklığında inkübe edin. (NOT: SSEA-4 boyama, doğrudan 6. adıma gidin) </li><li> 1xPBS, pH 7.4 ile üç kez durulayın FITC-konjuge tavşan IgG (% 0,2 BSA 1:500 dilüsyon), ve 1 saat oda sıcaklığında inkübe ekleyin. </li><li> 1xPBS, pH 7.4 ile 3 kez durulayın ve iyi merkezi montaj çözümü bir damla koymak (nükleer görselleştirme isteniyorsa DAPI içerebilir). Oje mühür, hücrelerin üstüne lamel koyun, ve immünofloresan mikroskobu ile değerlendirmek. </li></ol> İnsan embriyonik kök hücre makbuzlar ES medya (Embriyonik Kök hücre ortam): DMEM/F12 - 400ml Nakavt Serum Replacer - 100ml Non-Essential Amino Asitler - 5ml 200mm GlutaMax / BME Çözüm - 5ml Penisilin / Streptomisin - 5ml 4 iyice karıştırınız ve 500ml şişelerde son hESC Kültür Medya saklamak ° C En fazla bir iki hafta iyi. MEF Kültür Medya: DMEM - 1000ml FBS - 100ml Non-Essential Amino Asitler - 10ml Glutamin - 10ml Penicllin / Streptomisin - 10ml İyice karıştırın ve 4 final MEF Kültür Medya saklamak ° C. 200mm Glutamax / BME Çözüm: Glutamax - 100ml β-mercaptoethanol (14.3M Stok çözelti) - 140 μ1 Iki ve sonra çözümler kısım 10ml bölümlerini karıştırın. -20 ° C&#39;de alikotları tutun bFGF Çözümü (stok nihai konsantrasyonu: 10 mikrogram / ml): bFGF (FGF2) - 50 mikrogram 1xPBS% 0,1 BSA, pH: 7.4 - 5ml 10 mg / ml stok yapmak için 50 mikrogram olarak 5ml% 0.1 BSA bFGF eritin. 500 ul parçaya kısım. -80 ° C saklayınız Kollajenaz IV Çözümü (1mg/ml): Kollajenaz IV - 50mg DMEM/F12 Medya - 50ml 50ml şahin tüp kollajenaz IV tozu ekleyin. Kaput, 50ml steril DMEM/F12 medya karıştırın. &lt;1 dakika vorteksleyin çözüm. toz çözülmüş olduğundan emin olun. Kaputu, yeni bir steril 50ml şahin tüp içine filtresi 0.22 mikron geçmesi. Bu çözüm için iyi saklanan 2 hafta, 4 ° C maksimum Dispase Çözüm (1mg/ml): Dispase 5mg/ml - 2ml DMEM/F12 Medya - 8 ml -20 ° C&#39;de kısım ticari, 5mg/ml Dispase çözüm ve mağaza DMEM/F12 kullanarak 1mg/ml dispase sulandırınız. Bu çözüm, 1 hafta, 4 tutulmalıdır ° C.

<ol>
	<li>Thomson, J. a. m. e. s. A., Itskovitz-Eldor, J. o. s. e. p. h., Shapiro, S. a. n. d. e. r. S., Waknitz, M. i. c. h. e. l. l. e. A., Swiergiel, J. e. n. n. i. f. e. r. J., Marshall, V. i. v. i. e. n. n. e. S., Jones, J. e. f. f. r. e. y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+Stem+Cell+Lines+Derived+from+Human+Blastocysts+.">Embryonic Stem Cell Lines Derived from Human Blastocysts .</a> Science. 282, 1145-1147 (1998).</li><li>Xu, C. h. u. n. h. u. i., Inokuma, M. a. r. g. a. r. e. t. S., Denham, J. e. r. r. o. d., Golds, K. a. t. h. a. l. e. e. n., Kundu, P. r. a. t. i. m. a., Gold, J. o. s. e. p. h. D., Carpenter, M. e. l. i. s. s. a. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells+Nature+Biotechnology+19."> Feeder-free growth of undifferentiated human embryonic stem cells Nature Biotechnology 19.</a> Nature Biotechnology. 19, 971-974 (2001).</li></ol>

Bu dizi 3 video (MEF) besleyici hücreler (video 1), geçit onları besleyici hücre-levhalar (Video 2) Matrigel nasıl ve ne kadar korumak için fare embriyonik fibroblast insan embriyonik kök hücreleri (hESC) büyümeye nasıl gösterir Matrigel besleyici hücre koşulları (video 3) Pasajlanması hESC. Birçok önceki çalışmalar, bu bakım yaşayabilir, farklılaşmamış hESC inaktive MEF besleyici hücreler kültür gerektirir gösterdi. Ancak, pek çok deney, besleyici hücre kontaminasyon ücretsiz hESC saf bir nüfus gerekmektedir. Bu amaca ulaşmak için, biz nasıl geçit hESC MEF besleyici plakaları besleyici hücre ücretsiz Matrigel plakaları ve nasıl korumak ve besleyici sürekli geçit hESC kolonilerin hücre koşullarda göstermiştir. MEF besleyici hücre kontaminasyon bu protokolü takip ederek, küçük bir miktar hala bir Matrigel plaka üzerinde geçiş birine de var olabileceği, ancak bu kirletici genellikle görselleştirmek için kolaydır. Matrigel geçit iki ötesinde, hiçbir MEF kirlenme genellikle deneyler için saf bir nüfus hESC bırakarak bulundu. Ekim-4 ve SSEA-4, hESC pluripotency belirteçleri, immünofloresan boyaması ve mikroskobu veya flow sitometri sırasında farklılaşmamış bir kültür devlet hESC bakım onaylamak için ihtiyaç vardır.

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Knockout Serum Replacer (KSR)</td>
<td>Reagent</td>
<td>Gibco</td>
<td>10828-028</td>
<td>&nbsp;</td>
<tr>
<td>DMEM/F12</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11330-057</td>
<td>&nbsp;</td>
<tr>
<td> Non-essential Amino Acids</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11140-050</td>
<td>&nbsp;</td>
<tr>
<td>GlutaMax </td>
<td>Reagent</td>
<td>Gibco</td>
<td>35050-061</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11995-065</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>Reagent</td>
<td>Clontech</td>
<td>631107</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>Reagent</td>
<td>Gibco</td>
<td>25030-081</td>
<td>&nbsp;</td>
<tr>
<td>BME</td>
<td>Reagent</td>
<td>Fisher</td>
<td>BP176-100</td>
<td>&nbsp;</td>
<tr>
<td>bFGF</td>
<td>Reagent</td>
<td>R&D Systems</td>
<td>233-FB-025</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase IV</td>
<td>Reagent</td>
<td>Gibco</td>
<td>17104-019</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>Reagent</td>
<td>Stem Cell Technologies</td>
<td>17105-041</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin / Streptomycin </td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Gelatin</td>
<td>Reagent</td>
<td>Chemicon</td>
<td>ES-006-B</td>
<td>&nbsp;</td>
<tr>
<td>Matrigel</td>
<td>Reagent</td>
<td>BD Biosciences</td>
<td>354277</td>
<td>&nbsp;</td>
<tr>
<td>Oct-4 antibody</td>
<td>Reagent</td>
<td>Santa Cruz Biotechnology</td>
<td>SC-9081</td>
<td>&nbsp;</td>
<tr>
<td>anti-h/mSSEA-4 Phycoerythrin Conjugated Mouse IgG3</td>
<td>Reagent</td>
<td>R&D Systems</td>
<td>FAB1435P</td>
<td>&nbsp;</td>
<tr>
<td>FITCI-conjugated antirabbit IgG</td>
<td>Reagent</td>
<td>Jackson ImmunoResearch Laboratories. Inc.</td>
<td>715-095-152</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
					</div>

from mefs to matrigel 3: passaging hescs from matrigel onto matrigel

Bu video, besleyici, hücre koşullarda ve ne kadar besleyici sürekli geçit hESC hücre koşulları, insan embriyonik kök hücreleri (hESC) büyümesini nasıl koruyacağınızı gösteriyor. HESC, Onay pluripotency immünofloresan mikroskobu ile besleyici, hücre koşullarda yetiştirilen da gösterilmiştir.

Watch this Scientific Journal Video about From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel at JoVE.com

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

Bu video, besleyici, hücre koşullarda ve ne kadar besleyici sürekli geçit hESC hücre koşulları, insan embriyonik kök hücreleri (hESC) büyümesini nasıl koruyacağınızı gösteriyor. HESC, Onay pluripotency immünofloresan mikroskobu ile besleyici, hücre koşullarda yetiştirilen da gösterilmiştir. 3 Bölüm 3

Matrigel MEFS Matrigel 3: Matrigel üzerine gelen Pasajlanması hESC

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage ...

from-mefs-to-matrigel-3-passaging-hescs-from-matrigel-onto-matrigel

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Biology

13.7K Görüntüleme.  University of California, Los Angeles. Bu video, besleyici, hücre koşullarda ve ne kadar besleyici sürekli geçit hESC hücre koşulları, insan embriyonik kök hücreleri (hESC) büyümesini nasıl koruyacağınızı gösteriyor. HESC, Onay pluripotency immünofloresan mikroskobu ile besleyici, hücre koşullarda yetiştirilen da gösterilmiştir. 3 Bölüm 3

Video: From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

Tripsin ile Pasajlanması tonlar İnsan Embriyonik Kök Hücre hatları

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

Biyoloji

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

İnsan Nöral Kök Hücreler Pasajlanması

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.


This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. Part 1 of 3.

MEFS I Matrigel: MEFS Durum Pasajlanması hESC

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.
...

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

Sox-2 Zorla İfade yoluyla MEFS iPS Hücreler oluşturma, Eki 4, c-Myc ve Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF plates to feeder cell-free Matrigel plates.


This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.

MEFS Matrigel 2 Matrigel üzerine MEFS Yarma hESC:

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF ...

Cargo Loading onto Kinesin Powered Molecular Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport.
These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles 1. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available.
Building on the classic inverted motility assay 2, the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo 3. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface.
The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA4, quantum dots 5 or a wide variety of antigens via biotinylated antibodies 4-6.

Molecular shuttles consisting of functionalized microtubules gliding on surface-adhered kinesin motor proteins can serve as a nanoscale transport system. Here, the assembly of a typical shuttle system is described.

Kinesin Powered Moleküler minibüsleri üzerine Kargo Yükleniyor

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport ...

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

Solucan Lab An Introduction to: Kültürleme Worms Mutagenez

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Tümör Hücrelerinin vaskülojenik Faaliyet değerlendirin bir Matrigel Tabanlı Tüp Oluşumu Testi

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Verimli Üretimi ve Saflaştırılması Rekombinant murin kindlin-3 Biophysical Çalışmaları Böcek hücrelerinden

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Epigenetik yeniden programlanması ile hESC itibaren Myospheres Üretimi

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...