Yazarlar, Çocuk Hastanesi, Emory Üniversitesi&#39;nde hNSPC kültürler ve Dr. Gary Bassell parafilm boyama tekniği ilk öğretim sağlamak için Orange County Araştırma Enstitüsü Ulusal İnsan Nöral Kök Hücre Kaynak Dr. Philip H. Schwartz kabul etmek istiyorum.

1. Gün: Alman Cam lameller hazırlanması ve Seeding Hücreler Not: Alman cam lamelleri genellikle kültür birincil sinir kök hücreleri ve nöronlar için tercih edilir. Biz hücre yapışması ve yayılmasını geliştirmek için aşağıdaki temizleme protokolünü kullanır. Lamel her iki taraf için sıvı erişim sağlayacaktır küçük bir raf pozisyon lamelleri. İlk olarak, 10 dakika boyunca% 1 Liquinox inkübe lamelleri, deiyonize su ile 3 yıkar. Sabun köpükleri kalır emin olun. Sonra, 30 dakika boyunca inkübe fişleri 1M HCl, deiyonize su ile 3 yıkar. Kuru lamelleri 65 ° C geceleme. Sterilize etmek için bir bardak çanak ve otoklav lamelleri aktarın. <ol><li> Bir doku kültürü kaputu, uygun büyüklükte bir plaka steril lamelleri yerleştirin. </li><li> Hücre adezyon laminin ile Coat lamelleri. 10 mg / 5 dakika oda sıcaklığında ml steril su poli D-Lizin (PDL) inkübe edin lamelleri. 37 ° C doku kültürü inkübatör, bir gecede 4 saat, ya da için EMEM de 20 mikrogram / ml laminin PDL ve inkübe lamelleri çıkarın. <ol><li> Not: Pasajlanması İnsan Nöral Kök Hücreler makale (bakın <a href="/index/Details.stp?ID=263">https://www.jove.com/index/Details.stp?ID=263</a> ) ve Sayım İnsan Nöral Kök Hücreler makale ( <a href="/index/Details.stp?ID=262">https://www.jove.com / index / Details.stp? ID = 262</a> hNSPCs tekrar süspansiyon ve saymak nasıl öğrenmek için). </li></ol></li><li> Her lamel içeren belirli bir yoğunluk (genellikle immün için kaplama olduğunu hücreler için 100,000-300,000 hücre / ml &#39;lik bir başlangıç ​​kaplama yoğunluğu), laminin PBS ve tohum hücreleri ile kaplı lamelleri durulayın. </li><li> 37 Place plaka hücreleri lamelleri (genellikle gerekli olan en az 4 saat) bağlı ° C doku kültürü kuvöz ve izin verir. </li></ol> 2. Gün: Sabitleme, Permeabilizing, engelleme ve Hücreler İlköğretim Antikor ekleme Not: Biz hücre morfolojisi korumak için aşağıdaki% 4 paraformaldehid fiksatif kullanın. Tarifi paraformaldehid çözüm içine gitmek için izin pH geçiş içerir. Formik asit, floresan kullanırken arka plan boyama artırabilir nesil yüksek sıcaklıklarda yol açabilir, çünkü bu yöntem yerine paraformaldehid çözmek için ısı kullanımı tercih ederler. Diğerleri genel morfolojisi (sukroz) korumak için yardım ederken fiksatif yardım bazı bileşenleri, iskelet yapısı (MgCl 2 ve EGTA) korumak için. Hücreler için% 4 Paraformaldehit Sabitleme <table style="border-collapse: collapse; border: medium none" border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Reaktifler ve adımları izleyin: </td><td> 100 ml için sabitleştirici Miktarı: </td></tr><tr><td> MilliQ su </td><td> 75 ml </td></tr><tr><td> Paraformaldehit (davlumbaz dışarı tartmak) </td><td> 4 g </td></tr><tr><td> 10N NaOH, karıştırın ve çözüm netleşene kadar damla damla ekleyin </td><td> gerektiği gibi </td></tr><tr><td> 10X PBS </td><td> 10 ml </td></tr><tr><td> 1M MgCl 2 </td><td> 0.5 ml </td></tr><tr><td> 0.5 M EGTA </td><td> 2 ml </td></tr><tr><td> Sakaroz </td><td> 4 g </td></tr><tr><td> 6N HCl, pH 7.4 ile titre </td><td> gerektiği gibi </td></tr><tr><td> MilliQ su </td><td> 100 ml getirmek </td></tr><tr><td colspan="2"> 1 hafta kadar 4 ° C&#39;de saklayın. 37 sıcak ° C kullanmadan önce </td></tr></tbody></table><ol><li> Bankta, medyayı çıkartın ve prewarmed fiksatif eklemek. 5-15 dakika (antijen tespit edilmesi bağlı olarak, biz genellikle 10 dakika) inkübe edin. Bu ve oda sıcaklığında aşağıdaki adımları uygulayın. </li><li> Pek çok kurum, tehlikeli atık olarak paraformaldehid sapı gibi, uygun bir kap içine fiksatif atın. Hücreleri PBS ile 3 kez, 5 dakika, her zaman yıkayınız. Hücrelerin çözüm çıkarırken, emme hücrelerin kurumasına olmadığını dikkatli olun. Ayrıca, her zaman kat onları böylece hücreleri kurumaya bırakılır değildir ki çözüm çıkarmadan önce hücre eklemek yandan sonraki çözüm var. </li><li> Ilgi antijen, hücre zarı, hücre içinde antikor girişine izin geçirgen hale gerekir ise. Hücreler permeabilize için, PBS içinde taze% 0.3 Triton X-100 çözümü kullanın. Pipet yavaş Triton viskoz, ve deterjanın tamamen hücrelere eklemeden önce PBS içinde çözülür emin olun çünkü. PBS, 5 dakika her zaman hücreleri 3 kez yıkanarak 5 dakika hücreleri, Permeabilize. </li><li> Hücreler non-spesifik antikor bağlanma önlemek için engelleyici bir ajan ile tedavi edilmelidir. Biz (BSA) engelleyici bir ajan olarak sığır serum albumin kullanın. (Bunu çözmek için zaman alır, çünkü bir rocker veya rotator) engelleme çözüm PBS% 5 BSA eriterek sonra sol filtreleme0.45 mikron şırınga filtresi ile ution. Oda sıcaklığında 1 saat süreyle% 5 BSA / PBS çözüm hücrelerin engelleyin. </li></ol> Primer antikor (umarım özel ilgi protein)% 1 önceden belirlenmiş bir konsantrasyona seyreltilir BSA / PBS (% 5 BSA / PBS seyreltme tarafından hazırlanan). Yeri parafilm ile kaplanmış bir cam plaka üzerinde birincil antikor (25 mm lamel başına 30 ul, 18 mm lamel başına 20 ul, 12 mm lamel başına 15 ul) seyreltilir. Hücre aşağı yüz, ve böylece hücrelerin antikor ile temas antikor çözüm üzerinde yattığını, invert sabit hücreleri ile lamel Pick up. Tüm yapı üzerinde ikinci bir parafilm şeridi yerleştirin. 4 ° C gece boyunca inkübe edin. 3. Gün: Yıkama, İkincil Antikor Kuluçka, Nükleer Leke ve Montaj <ol><li> Dikkatlice hücreler yukarı bakacak şekilde üst parafilm şerit kaldırmak ve lamel pick up, ters ve her iyi yıkama için PBS ile plaka içine geri koyun. </li><li> Hücreleri PBS ile 3 kez, 5 dakika, her zaman yıkayınız. </li><li> Birincil antikor tespit birincil antikor bir tavşan yapıldı örneğin, ikincil antikor tavşan IgG tanımalıdır ikincil bir antikor seçin. (IgG, IgM, vb.) Antikor izotip maç için özen gösterin. Multipl primer antikorlar kullanılarak, farklı türler veya isotypes ve ikincil antikorlar bağlı farklı belirleyicileri ile tespit etmek için planı emin olun. Örneğin, yeşil bir fluorofor bir anti-fare ikincil birleştiğinde kırmızı fluorofor bir anti-tavşan ikincil birleştiğinde kullanabilirsiniz. Ilköğretim için aynı tekniği kullanarak, karanlıkta, oda sıcaklığında 2 saat için% 1 BSA uygun bir konsantrasyon seyreltilmiş ikincil antikor çözüm hücreleri inkübe (video gösterilmeyen adımlar). Ikincil bir gliserol solüsyonu (25 mm lamel başına 40 ul, 18 mm lamel başına 30 ul, 12 mm lamel başına 25 ul) olarak saklanır, kuluçka için gerekli ikincil antikor hacmi birincil antikor için biraz daha büyük olabilir . <ul><li> Not: floresan moleküller ikincil antikor görselleştirmek için kullanıyorsanız, örnek ikincil antikor eklenmiştir sonra ışıktan korunması gerekir. Karanlık, ya da folyo kaplı plaka inkübe edin. </li></ul></li><li> Primer antikor açıklandığı gibi parafilm lamelleri dikkatlice çıkarın. Hücreleri, 2 mcg / ml Hoechst nükleer PBS içinde seyreltilmesi leke (bir nükleer counterstain tercih edilir) bir 1 dakika inkübasyon PBS ile 2 kez, her zaman 5 dakika yıkayınız. Hoechst leke eski ve sinyal çok zayıfsa, inkübasyon süresi ve / veya konsantrasyonu artırabilir. Hoechst ile inkübasyondan sonra, 5 dakika boyunca 1 kez PBS ile yıkayın. </li><li> Lamelleri mikroskop görünüm için montaj medya ile bir slayt monte edilmelidir. Ikincil antikor görselleştirme için kullanılan floresan molekül olduğu durumlarda, montaj medya photobleaching en aza indirmek için ajan içermelidir. Biz, floresan bir montaj medya olarak Vectashield kullanın. Bir mikroskop lamı (25 mm lamel başına 12 ul, 18 mm lamel başına 6 ul, 12 mm lamel başına 3 ul) Vectashield uygun bir boyut damla yerleştirin. </li><li> Lamel dikkatlice pick up ve arka tuzları (PBS) kaldırmak için deiyonize su ile durulayın. Kimwipe veya kağıt havlu üzerine aşırı su kapalı Dab ve aşağı bakacak hücreleri ile Vectashield açılan yatıyordu. Lamel bir açıyla yerleştirin ve yakalama hava kabarcıkları önlemek için yavaş yavaş inmeye izin. Tarih ve herhangi bir örnek bilgileri slayt etiketleyin. </li><li> Lamelleri ile slaytlar lamel kenarı etrafında (koyu) aşırı Vectashield kapalı sonra emme kurumasını bekleyin. Istenirse lamel kenarları (özellikle yararlıdır lamelleri ters bir mikroskopta görülebilir.), Oje ile kapatılmış olabilir. Biz genellikle, bizim slaytlar -20 ° C derin dondurucuda saklayın. </li></ol>

<ol>
	<li>Flanagan, L. A., Chou, J., Falet, H., Neujahr, R., Hartwig, J. H., Stossel, T. P., Filamin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamin+A,+the+Arp2/3+complex,+and+the+morphology+and+function+of+cortical+actin+filaments+in+human+melanoma+cells.">Filamin A, the Arp2/3 complex, and the morphology and function of cortical actin filaments in human melanoma cells.</a> J. Cell Biol. 155, 511-518 (2001).</li><li>Flanagan, L. A., Rebaza, L. M., Derzic, S., Schwartz, P. H., Monuki, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+human+neural+precursor+cells+by+laminin+and+integrins.">Regulation of human neural precursor cells by laminin and integrins.</a> J. Neurosci. Res. 83, 845-856 (2006).</li><li>Flanagan, L. A., Ziaeian, B., Palmer, T., Schwartz, P. H., Loring, J. F., Wesselschmidt, R. W., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+analysis+of+stem+cells.">Immunocytochemical analysis of stem cells.</a> In Human Stem Cell Manual: A Laboratory Guide. , (2007).</li><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passaging+Human+Neural+Stem+Cells.">Passaging Human Neural Stem Cells.</a> Journal of Visualized Experiments. 7, (2007).</li><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+Human+Neural+Stem+Cells.">Counting Human Neural Stem Cells.</a> Journal of Visualized Experiments. 7, (2007).</li></ol>

Bu protokol, gerekli antikor hacmi en aza indirir ve güvenilir hücre boyama verir hNSPCs bir immün prosedürü anlatılmaktadır. Açıklandığı gibi işlem hücre içi antijenler için iyi, ama hücre yüzey molekülleri leke veya hücre iskeletinin boyama artırmak için modifiye edilebilir.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>German glass (Deutsche Spiegelglas) coverslips</td>
<td>Tool</td>
<td>Carolina Biological Supply Company</td>
<td>WW-63-3029</td>
<td>12,15,18, 22, 25 mm coverslips available</td>
</tr>
<tr>
<td>Liquinox phosphate-free detergent</td>
<td>Reagent</td>
<td>Sigma</td>
<td>Z273279</td>
<td>very viscous</td>
</tr>
<tr>
<td>Poly-D-lysine hydrobromide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P7280</td>
<td>&nbsp;</td>
<tr>
<td>Laminin, natural mouse</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>23017-015</td>
<td>&nbsp;</td>
<tr>
<td>EMEM (1X)</td>
<td>Reagent</td>
<td>Mediatech</td>
<td>MT-10-010-CV</td>
<td>Distributed by Fisher under the indicated catalog #</td>
</tr>
<tr>
<td>24 well plate</td>
<td>Tool</td>
<td>Fisher</td>
<td>08-772-1</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Fisher</td>
<td>BP151-500</td>
<td>very viscous</td>
</tr>
<tr>
<td>Paraformaldehyde</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P6148</td>
<td>potential carcinogen, use caution when using it in powder and solution form</td>
</tr>
<tr>
<td>Bovine Serum Albumin, IgG-free</td>
<td>Reagent</td>
<td>Jackson Immunoresearch</td>
<td>001-000-161</td>
<td>&nbsp;</td>
<tr>
<td>Hoechst 33342</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>H-1399</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield</td>
<td>Reagent</td>
<td>Vector Laboratories</td>
<td>H-1000</td>
<td>viscous</td>
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immunocytochemistry: human neural stem cells

İmmünositokimya olup olmadığının belirlenmesi için çok güçlü ve oldukça basit bir yöntemi, hücre içi lokalizasyonu ve faiz, en yaygın kültür hücrelerinde protein, antijen göreceli bolluk. Bu protokol sunan, kolay takip onların boyama, yüksek kalite, tekrarlanabilir nitel ve nicel veri alırken araştırmacılar primer ve sekonder antikorlar tasarrufu sağlayacak adımları bir dizi. Bu protokolün boyanması için gerekli antikor hacminin korunmasına yardımcı iki yönü vardır. Biri için, hücrelerin doku kültürü plaka kuyulara yerleştirilen küçük, yuvarlak lamelleri yetiştirilmektedir. Fiksasyon sonra, lamelleri hücreleri plaka kuyulardan silinebilir. Antikor boyama, hücre ile lamel parafilm antikor çözüm küçük bir damla üzerine ters ve parafilm kurumasını önlemek için ikinci bir parça ile kaplıdır. Bu yöntemi kullanarak, sadece antikor çözüm ~ 25 ul boyanmış her lamel (ya da örnek) için gereklidir. Bu protokol, insan sinir kök / prekürsör hücrelerinin (hNSPCs) immün açıklar, ama birçok diğer hücre türleri için kullanılabilir.

Watch this Scientific Journal Video about Immunocytochemistry: Human Neural Stem Cells at JoVE.com

Immunocytochemistry: Human Neural Stem Cells

İmmünositokimya varlığı, hücre içi lokalizasyonu ve kültür hücreleri ilgi antijen göreceli bolluk belirlemek için güçlü bir yöntemdir. Bu protokol sunan, kolay takip serisi bir antikorlar korunması ve boyama en iyi şekilde almak için sağlayacak adımlar.

İmmünositokimya: İnsan Sinir Kök Hücreler

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of ...

immunocytochemistry-human-neural-stem-cells

Research

JoVE Journal

Biology

27.2K Görüntüleme.  University of California, Irvine (UCI). İmmünositokimya varlığı, hücre içi lokalizasyonu ve kültür hücreleri ilgi antijen göreceli bolluk belirlemek için güçlü bir yöntemdir. Bu protokol sunan, kolay takip serisi bir antikorlar korunması ve boyama en iyi şekilde almak için sağlayacak adımlar.

Video: Immunocytochemistry: Human Neural Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

İnsan Embriyonik Kök Hücre (ES) yayılması

Biyoloji

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

Amaxa Nucleofector İnsan Sinir Kök Hücreler Transfecting

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

Counting Human Neural Stem Cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

İnsan Nöral Kök Hücreler Sayma

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

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 ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Immunosurgery İnsan Embriyonik Kök Hücreleri türetilmesi

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

Fare Epidermal Sinir Crest Kök Hücre (EPI-NCSC) Kültürler

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

İnsan Embriyonik Kök Hücre Kromatin Immunoprecipitation

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

İnsan CD40 aktive B hücreleri üretilmesi

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

İnsan Embriyonik Kök Hücreler Donma ve Çözülme

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Embriyonik Kök Hücreler farklılaşma oligodendrosit Öncüleri

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...