Anyaporn Sawasdichai ve Nazefah Abdülhamit&#39;in doktora sırasıyla Kraliyet Tayland Hükümeti ve Malezya Hükümeti minnettarız Burslar. Bu çalışma, Wellcome Trust PSJ ve KG için verilen bir proje hibe tarafından finanse edildi. Ayrıca, Bristol Wolfson bio Tesisi Üniversitesi minnettarız.

I. fraksiyonasyon için hazırlanması Bu bölümde, poli-L-Lizin kaplı mikroskop lamelleri ve fraksiyonasyon önce hücre eki hazırlama açıklamaktadır. Hücrelerin Gerekirse geçici eki önce veya sonra ya protein ekspresyon vektörleri ile transfekte olabilir. A. poli-L-Lizin kaplı lamelleri hazırlanması <ol><li> Distile su 1mg/ml poli-L-Lizin bir çözüm hazırlayın. </li><li> Sallanan bir platform üzerinde en az 1 saat için 22 çözüm onları kuluçka poli-L-Lizin Coat temiz lamelleri ° C </li><li> Steril distile su ile kaplı lamelleri iki kez yıkayın ve% 96 etanol ile tek bir yıkama ile takip. </li><li> Hava filtre kağıt parçası üzerinde kaplı lamelleri kuru ve kuru bir konteyner (bir kez kuru onlar yığılmış olabilir) ileride kullanmak üzere saklayın. </li></ol> Lamelleri B. takılması hücreleri Sigara yapışık hücrelere (burada K562 hücreleri kullanır) <ol><li> 6-plaka kaplı yüzeyi yukarı bakacak şekilde çok iyi bir poli-L-Lizin kaplı lamel yerleştirin. </li><li> 7x10 5 hücre yoğunluğu Tohum K562 hücre / Dulbecco&#39;nun Modifiye Eagles Medium (DMEM)% 10 fetal sığır serumu (FBS), PS (penisilin 100units/ml, streptomisin 100mg/ml) ile desteklenebilir. K562 hücrelerinin durumda geçici transfeksiyon hücrelerin ekim önce elektroporasyon (0.4cm elektroporasyon küvetler 250V / 975μF medya 200μl 1x10 7 hücre) kullanılarak yapılabilir. </li><li> 37 ° C% 5 CO 2 hücreleri az 24 saat süreyle inkübe edin. </li><li> Orta kapalı dökün ve hücreler iki kez yıkayın buz fosfat tamponlu salin (PBS). </li><li> Subselüler fraksiyonasyon protokolü ile izleyin. </li></ol> Yapışık hücrelere (burada Cos-7 hücre) <ol><li> 6 plaka iyi bir kaplanmamış lamel yerleştirin. </li><li> 37 ° C PBS içinde iki kez yıkayın prewarmed </li><li> , 37 tripsin (% 0.03 EDTA,% 0.25 &#39;lik tripsin) PBS içinde sindirerek hücrelerin Ayır ° C&#39;de 2 ila 5 dakika. Bu ağırlıklı olarak bireysel yüzen hücreler gibi hücrelerin aşırı sindirimi ve sindirim en kısa sürede durdurmak için önemlidir. </li><li> /% 10 FBS ve PSG (penisilin 100 ünite / ml, streptomisin 100ug/ml ve L-glutamin 292ug/ml) ile desteklenmiş DMEM 6 plaka ekim 3x10 5 hücre yoğunluğu hücreler tarafından sindirimi durdurur normal lamelleri. Yapışık hücrelere genellikle gerekirse Ancak, poli-L-Lizin kaplı lamelleri kullanılabilir, normal mikroskop lamelleri üzerine takın. </li><li> 37 ° C ve% 5 CO 2 hücreleri az 24 saat süreyle inkübe edin. Cos-7 hücreleri durumda geçici transfeksiyon Fugene gerekirse 6 (Roche) ve 37, bir 24 saat daha kurtarmak için sol hücreleri kullanılarak yapılabilir ° C ve% 5 CO 2 . </li><li> Orta dökün ve hücrelerin buz PBS ile iki kez yıkayın. </li><li> Subselüler fraksiyonasyon protokolü ile izleyin. </li></ol> II. Subselüler fraksiyonasyon In situ fraksiyonu Şekil 1&#39;de gösterildiği gibi şematik göre yapılır ve bir yönteme dayalı daha önce 5 açıklanan değişiklikler, daha önce 7 açıklanan ve aşağıda ayrıntılı bir protokol. Bu sıvı çıkarmadan önce lamel çıkarmadan her fraksiyonasyon adım izleyerek yeni bir 6-plaka lamelleri aktarmak için tavsiye edilir. Sadece dört lamelleri görüntüleme için gerekli olmasına rağmen, ek lamelleri, bu paralel olarak yapılacak ise batı blot tam hücre özü ve nükleer matriks kesirler üretmek için gereklidir. <ol><li> Hazırlayın ve hücre iskeletinin tampon (CSK) filtresi: 10mM BORU, 300mm Sakkaroz, 100 mM NaCl, 3mm MgCl 2, 1mm EGTA. CSK tampon fraksiyonasyon gün taze hazırlanmalıdır. </li><li> Gerekirse bir lamel hücreleri doğrudan lamel üzerine hafifçe 200ul TES tampon (% 1 SDS, 2mm EDTA, 20mm Tris-HCl pH: 7.4) yerleştirerek ve 1 ya da 2 dakika süreyle inkübe blot batı için bütün hücre elde etmek için kullanılabilir 22 ° C Sonra 1M 250ul (NH 4) 2 SO 4 ve diğer batı örnekleri hazır olana kadar buz üzerinde inkübe ekleyerek tüm hücre özü çıkarılır ve proteinleri presipite olabilir. </li><li> 22 ° C eğerek 6 iyi plakalı iki veya üç kez kalan lamelleri, 1 ml buz PBS ile iki kez yıkayın. </li><li> Tüm hücre ve taze bir 6-plaka transfer temsil eden lamel çıkarın. Hücre tespit ve immuno-floresans protokol ile izleyin. </li><li> Kalan kuyulardan PBS yavaşça çıkarın. </li><li> Yavaşça 200ul CSK tampon yerleştirerek, sitoplazmik ve gevşek düzenlenen nükleer proteinlerin Özü + 0.1% (V / V) doğrudan kalan her lamel üzerine Triton X-100 ve kuluçka1 dakika boyunca buz. </li><li> Sitoplazmik ve gevşek tutulan nükleer özü çıkarın ve 2. adımda açıklandığı gibi hızlandırabilir. </li><li> 22 lamelleri 1ml buz PBS ile iki kez yıkayın ° C devirme 6 plakalı. </li><li> Sıkıca tutulan nükleer malzeme temsil lamel çıkarın ve hücre tespit ve immuno-floresans protokol ile takip. </li><li> Sonra yavaşça geri kalan kuyulardan PBS kaldırmak + 0.5% (V / V) Triton X-100 doğrudan kalan her lamel üzerine hafifçe 200ul CSK tampon yerleştirerek ve 20 dakika boyunca buz üzerinde inkübe tarafından sıkıca tutulan proteinler ayıklayın. </li><li> Sıkıca tutulan özü çıkarın ve 2. adımda açıklandığı gibi hızlandırabilir. </li><li> 22 lamelleri 1ml buz PBS ile iki kez yıkayın ° C devirme 6 plakalı. </li><li> Kromatin fraksiyonu temsil eden lamel çıkarın ve hücre fiksasyon ve immüno-floresans protokol ile takip. </li><li> CSK sonra yer 200ul tamponu + 100ug/ml DNaz I 30 dakika kalan lamelleri ve inkübe üzerine 37 ° C yavaşça kalan kuyulardan PBS kaldırmak </li><li> Kromatin özü çıkarın ve 2. adımda açıklandığı gibi hızlandırabilir. </li><li> ° C eğerek 6-plaka 22 lamelleri buz PBS ile iki kez yıkayın. </li><li> Nükleer matriks fraksiyonu temsil lamel çıkarın ve hücre tespit ve immuno-floresans protokol ile takip. </li><li> Gerekirse matriks proteinlerine 2. adımda açıklandığı gibi 200ul TES tampon kullanarak blot batı için ek bir matris lamel çıkarılabilir. </li><li> Western blot örnekleri, 4 masa üstü mikrosantrifüj 13000 rpm&#39;de santrifüje tabi tutulmalıdır ° C ve 40ul SDS yükleme tamponu (62.5mm Tris-HCl, pH 6.8,% 2 SDS (w / v), 50 mM DTT, yeniden süspanse pelet % 10 gliserol, SDS-PAGE ile analiz öncesi% 0.01 Bromophenol Mavi (w / v)). <img src="/files/ftp_upload/1958/1958fig1.jpg" alt="Şekil 1" /> Şekil 1. 10mM BORU, 300mm Sakkaroz, 100 mM NaCl, 3mm MgCl 2, 1mm EGTA yerinde Subselüler fraksiyonasyon şematik Sitoiskelet tampon (CSK) oluşur. TES tampon% 1 SDS, 2mm EDTA, 20mm Tris-HCl pH: 7.4 oluşur. </li></ol> III. Hücre fiksasyon ve immüno-floresan <ol><li> Yavaşça 30 dakika boyunca her 4 lamel ve inkübe ° C için% 4 formaldehit / PBS 1 ml ekleyin. </li><li> Her lamel 22 PBS 1ml 2-3 kez hafifçe ° C yıkayın </li><li> , 22 ° C&#39;de 10 dakika boyunca her lamel ve inkübe% 0,1 Triton X-100/PBS 400ul ekleyin. </li><li> Her lamel 22 PBS 1ml 2-3 kez hafifçe ° C yıkayın </li><li> Potansiyel arka plan boyama azaltmak için 20 dakika boyunca 22 ° C&#39;de% 3 Bovine Serum Albumin (BSA) / PBS ve inkübe 400ul ekleyin. </li><li> Her lamel 22 PBS 1ml 2-3 kez hafifçe ° C yıkayın </li><li> 1 saat süreyle 22 ° C&#39;de inkübe üzerine lamel ve PBS içinde birincil antikor Yeri 100ul. Birincil ve ikincil antikorlar gerekli konsantrasyonu PBS içinde seyreltilmelidir. Bu, kullanılan her bir antikor için optimize edilmesi gerekebilir. </li><li> Her lamel 22 PBS 1ml 2-3 kez hafifçe ° C yıkayın </li><li> 22 ° C uzakta 1 saat süreyle ışık, PBS ve inkübe ikincil antikor Yeri 200ul. </li><li> Her lamel 22 PBS 1ml 2-3 kez hafifçe ° C yıkayın </li><li> DAPI (4 &#39;,6-diamidino-2-phenylindole hidroklorür) içeren Vectashield montaj orta kullanarak cam bir mikroskop lamı üzerine her lamel (hücre aşağı) monte edin. </li><li> Bir kağıt havlu kullanarak lamel dört bir yanından gelen aşırı montaj orta silin ve slayt ışıktan en az 30 dakika için 22 ° C&#39;de inkübe edin. </li><li> Lamel, şeffaf akrilik tırnak cilası kullanarak cam slayt sabitleyin. </li><li> Slaytlar floresan mikroskobu ile görselleştirme önce ışıktan uzak tutun. </li></ol> IV. Temsilcisi sonuçları <img src="/files/ftp_upload/1958/1958fig2.jpg" alt="Şekil 2" /> Şekil 2. Olmayan transfekte COS-7 hücrelerin hücre içi fraksiyonasyon fraksiyonlara Cos-7 hücreleri formaldehit ile sabit ve DAPI içeren orta montaj ile tedavi takip TRITC için konjuge A / C antikorları kullanarak immunohistokimyasal α-tubulin, α-Histon H1 veya α-Lamin . Görüntüler Leica konfokal mikroskop kullanılarak 10x oküler lens ve 63x objektif lens ile elde edildi. Hücre çekirdeklerinin işaretleyici antikorlar ile boyanarak aynı alanda hücreleri TRITC bir filtre seti kullanılarak görüntülendi DAPI filtre seti kullanılarak görüntülendi. <img src="/files/ftp_upload/1958/1958fig3.jpg" alt="Şekil 3" /> Şekil 3. GFP-6E2 füzyon proteini nükleer matriks ile ilişkilidir. Insan papilloma virüsü (HPV) tip 6 erimiş yeşil floresan proteininin (GFP) oluşan bir füzyon proteini ifade Cos-7 hücre hücre içi fraksiyonasyonE2 proteininin (GFP-6E2). Transfekte hücreler GFP-6E2 lokalizasyonu GFP floresan üzerinden doğrudan görüntülenmiştir iken Hücre çekirdekleri DAPI boyama ile görüntülendi. Lamin A / C α-Lamin TRITC ve TRITC bir filtre seti konjuge A / C antikorları kullanarak görüntülendi. Birleştirme görüntüler GFP-6E2 protein bazı nükleer matriks (sağ alt panel) ile ilişkili olduğunu ortaya koymaktadır. Şekil 2&#39;de açıklandığı gibi görüntüler elde edildi.

<ol>
	<li>Donaldson, M. M., Boner, W., Morgan, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopBP1+regulates+human+papillomavirus+type+16+E2+interaction+with+chromatin.">TopBP1 regulates human papillomavirus type 16 E2 interaction with chromatin.</a> J Virol. 81, 4338-4342 (2007).</li><li>Javed, A., Guo, B., Hiebert, S., Choi, J. Y., Green, J., Zhao, S. C., Osborne, M. A., Stifani, S., Stein, J. L., Lian, J. B., van Wijnen, A. J., Stein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Groucho/TLE/R-esp+proteins+associate+with+the+nuclear+matrix+and+repress+RUNX+(CBF(alpha)/AML/PEBP2(alpha))+dependent+activation+of+tissue-specific+gene+transcription.">Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBF(alpha)/AML/PEBP2(alpha)) dependent activation of tissue-specific gene transcription.</a> J Cell Sci. 113, 2221-2231 (2000).</li><li>Kowalczyk, A. M., Roeder, G. E., Green, K., Stephens, D. J., Gaston, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+induction+or+inhibition+of+apoptosis+by+HPV+proteins.">Measuring the induction or inhibition of apoptosis by HPV proteins.</a> Methods Mol. Med. 119, 419-432 (2005).</li><li>McLarren, K. W., Theriault, F. M., Stifani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+with+the+nuclear+matrix+and+interaction+with+Groucho+and+RUNX+proteins+regulate+the+transcription+repression+activity+of+the+basic+helix+loop+helix+factor+Hes1.">Association with the nuclear matrix and interaction with Groucho and RUNX proteins regulate the transcription repression activity of the basic helix loop helix factor Hes1.</a> J Biol. Chem. 276, 1578-1584 (2001).</li><li>McNeil, S., Guo, B., Stein, J. L., Lian, J. B., Bushmeyer, S., Seto, E., Atchison, M. L., Penman, S., van Wijnen, A. J., Stein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+the+YY1+transcription+factor+to+the+nucleolus+and+the+nuclear+matrix+in+situ:+the+C-terminus+is+a+principal+determinant+for+nuclear+trafficking.">Targeting of the YY1 transcription factor to the nucleolus and the nuclear matrix in situ: the C-terminus is a principal determinant for nuclear trafficking.</a> J Cell Biochem. 68, 500-510 (1998).</li><li>Melan, M. A., Sluder, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redistribution+and+differential+extraction+of+soluble+proteins+in+permeabilized+cultured+cells.+Implications+for+immunofluorescence+microscopy.">Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Implications for immunofluorescence microscopy.</a> J Cell Sci. 101, 731-743 (1992).</li><li>Zou, N., Lin, B. Y., Duan, F., Lee, K. Y., Jin, G., Guan, R., Yao, G., Lefkowitz, E. J., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hinge+of+the+human+papillomavirus+type+11+E2+protein+contains+major+determinants+for+nuclear+localization+and+nuclear+matrix+association.">The hinge of the human papillomavirus type 11 E2 protein contains major determinants for nuclear localization and nuclear matrix association.</a> J. Virol. 74, 3761-3770 (2000).</li></ol>

Yaygın sorunlar ve öneriler: Hücrelerin tamamı ya da bir çoğunu yıkama sırasında kaybolur. Yıkama adımları sırasında sıvı lamel kaçınarak 6 plaka yan yavaş yavaş ilave edilmelidir. Benzer şekilde, dikkatli bir şekilde devirme levha ile sıvı kaldırıldı olmalıdır ve aşırı sıvı yavaş yavaş pipetleme. Hücre adezyon poli-L-Lizin kaplı lamelleri kullanarak artırılabilir. Genomik DNA&#39;nın tamamen sindirilmiş değildir. Bazı hü...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>BSA</td>
<td>Chemical</td>
<td>Sigma</td>
<td>A9647-100G</td>
<td>&nbsp;</td>
<tr>
<td>DNase I</td>
<td>Chemical</td>
<td>Sigma</td>
<td>DN25-1G</td>
<td>&nbsp;</td>
<tr>
<td>poly-L-Lysine</td>
<td>Chemical</td>
<td>Sigma</td>
<td>P-8920</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>Chemical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>EGTA</td>
<td>Chemical</td>
<td>Sigma</td>
<td>E4378-25G </td>
<td>&nbsp;</td>
<tr>
<td>Fomaldehyde</td>
<td>Chemical</td>
<td>BDH</td>
<td>284216N</td>
<td>&nbsp;</td>
<tr>
<td>PIPES</td>
<td>Chemical</td>
<td>Sigma</td>
<td>P-6757</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>Chemical</td>
<td>Fisher Scientific</td>
<td>S/8600/53</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Chemical</td>
<td>Sigma</td>
<td>T-6876</td>
<td>&nbsp;</td>
<tr>
<td>Mounting Medium with DAPI</td>
<td>Chemical</td>
<td>Vector Laboratories</td>
<td>H-1200</td>
<td>&nbsp;</td>
<tr>
<td>Fugene 6 Transfection Reagent</td>
<td>Chemical</td>
<td>Roche </td>
<td>11814443001</td>
<td>&nbsp;</td>
<tr>
<td>Histone H1</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-8030</td>
<td>&nbsp;</td>
<tr>
<td>Lamin A/C</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-20681</td>
<td>&nbsp;</td>
<tr>
<td>Tubulin-&alpha;</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-32293</td>
<td>&nbsp;</td>		</tbody>
		</table>
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in situ subcellular fractionation of adherent and non-adherent mammalian cells

Protein fonksiyonu yakından protein lokalizasyonuna birleştirilmiştir. Yerinde Subselüler fraksiyonasyon bazı proteinler belirli bir konuma veya hücre içi bölmesi ile sınırlı olmasına rağmen, birçok protein, belirli bir Subselüler etki alanı veya yapısı ile sıkı sıkıya ilişkili bir alt nüfusa sahip, serbest döviz serbestçe difüzyon nüfus olarak mevcuttur. görselleştirme sağlar protein bölümlendirme ve aynı zamanda protein belirli yapılara lokalize alt-topluluklarda ortaya çıkarabilir. Örneğin, çözünür sitoplazmik proteinleri ve gevşek tutulan nükleer proteinlerin çıkarılması kromatin ile bazı transkripsiyon faktörleri istikrarlı bir ilişki ortaya çıkarabilir. DNA sonraki sindirim topluca nükleer iskele veya nükleer matriks denir protein ve RNA ağı ile birlikte, bazı durumlarda ortaya çıkarabilir. Burada, yapışık ve yapışmaz memeli hücrelerinin mikroskop lamelleri yerinde fraksiyonasyon sırasında gerekli adımları açıklar . Protein görselleştirme spesifik antikorların ya da floresan füzyon proteinleri ve floresan mikroskopi kullanılarak elde edilebilir. Antikorlar ve / veya özel bölme ya da yapılar için belirteç olarak hareket floresan boyalar protein yerelleştirme ayrıntılı olarak eşlenen izin yerinde fraksiyonasyon da batı her fraksiyon protein miktarları karşılaştırmak için blot ile kombine edilebilir. Bu basit biyokimyasal yaklaşım, aksi tespit edilmemiş kalacaktı dernekler ortaya çıkarabilir.

Watch this Scientific Journal Video about In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells at JoVE.com

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

, Memeli hücrelerinin mikroskop lamelleri yerinde Subselüler fraksiyonasyon protein localisation görselleştirme sağlar .

Yapışkan ve Sigara yapışık Memeli Hücre yerinde Subselüler Fraksiyonu

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

in-situ-subcellular-fractionation-adherent-non-adherent-mammalian

Research

JoVE Journal

Biology

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

15.6K Görüntüleme.  University of Bristol. , Memeli hücrelerinin mikroskop lamelleri yerinde Subselüler fraksiyonasyon protein localisation görselleştirme sağlar .

Video: In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Memeli Hücreleri Trypsinizing ve alt kültüre

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Biyoloji

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

Memeli Hücreleri RNAi Tarama MİSYON esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

İyon Kanalları ve Fonksiyonel Analiz Mutagenez Heterologously Memeli Hücreleri cinsinden ifade edilir

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Clonogenic Testi: yapışkan Hücreler

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Memeli Hücreler, Hücre Döngüsü Pozisyon Analizi

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

Bir genom Ölçeği Polizom Fraksiyonu ve Memeli Translatomes Analizi

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Memeli Hücrelerde Heme Sentezi Düzeylerinin Ölçümü

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Görüntüleme Yerel Ca<sup&gt; 2+</sup&gt; Kültürlenmiş memeli hücrelerinde sinyaller

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Dinamik Hücresel Protrüzyonlarda Protein Konsantrasyonunun Yazılım Destekli Takibi için Grafiksel Bir Kullanıcı Arayüzü

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

Otofajinin Kas Hücrelerinde Yerinde İmmunofloresan Boyanması

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...