The authors have no acknowledgements.

<ol>
	<li>Douek, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+thymic+function+with+age+and+during+the+treatment+of+HIV+infection.">Changes in thymic function with age and during the treatment of HIV infection.</a> Nature. 396 (6712), 690-695 (1998).</li><li>Zelm, M. C., Szczepanski, T., van der Burg, M., van Dongen, J. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+history+of+B+lymphocytes+reveals+homeostatic+proliferation+and+extensive+antigen-induced+B+cell+expansion.">Replication history of B lymphocytes reveals homeostatic proliferation and extensive antigen-induced B cell expansion.</a> J. Exp. Med. 204 (3), 645-655 (2007).</li><li>Fronkova, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-cell+reconstitution+after+allogeneic+SCT+impairs+minimal+residual+disease+monitoring+in+children+with+ALL.">B-cell reconstitution after allogeneic SCT impairs minimal residual disease monitoring in children with ALL.</a> Bone Marrow Transplant. 42 (3), 187-196 (2008).</li><li>Sottini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+quantification+of+recent+thymic+T-cell+and+bone+marrow+B-cell+emigrants+in+patients+with+primary+immunodeficiency+undergone+to+stem+cell+transplantation.">Simultaneous quantification of recent thymic T-cell and bone marrow B-cell emigrants in patients with primary immunodeficiency undergone to stem cell transplantation.</a> Clin. 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Neuroimmunol. 240-241, 147-150 (2011).</li><li>Zanotti, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+accumulation+of+newly+produced+T+and+B+lymphocytes+in+natalizumab-treated+multiple+sclerosis+patients.">Peripheral accumulation of newly produced T and B lymphocytes in natalizumab-treated multiple sclerosis patients.</a> Clin. Immunol. 145 (1), 19-26 (2012).</li><li>Sottini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-existing+T-+and+B-cell+defects+in+one+progressive+multifocal+leukoencephalopathy+patient.">Pre-existing T- and B-cell defects in one progressive multifocal leukoencephalopathy patient.</a> PLoS One. 7, e34493 (2012).</li><li>Quiros-Roldan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+combined+antiretroviral+therapy+on+B-+and+T-cell+release+from+production+sites+in+long-term+treated+HIV-1++patients.">Effects of combined antiretroviral therapy on B- and T-cell release from production sites in long-term treated HIV-1+ patients.</a> J. Transl. 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The authors have nothing to disclose.

TREC and KREC quantification can be considered a good estimate of recent thymic and bone marrow output provided that some caveats are taken into account. Even though an absolute quantification method employing standard curve requires more reagents and more space on the real-time PCR reaction plate, it ensures highly accurate quantitative results because unknown sample quantities are interpolated from standard curves built upon known amounts of starting material. Moreover this method is better fitted to detect low amount of targets. From the methodological point of view, a critical point is that the frozen plasmid dilutions corresponding to the standard curve points may degrade sooner than expected. This requires that new dilutions from the originally stored plasmid need to be prepared before each experimental procedure, in particular the most dilute ones. In the final calculations, a non-perfect equivalence between lymphocyte plus monocyte count as an estimate of the PBMC quantity may add some bias in the conversion between values expressed per 106 and values expressed per ml of blood. An improvement can be made to determine the amount of two targets (TRECs and KRECs) from a known amount of DNA extracted directly from whole blood, thus eliminating the need of the monocyte plus lymphocyte counts.14 In this specific case, a reference gene is not strictly necessary to perform the quantification, but may be used as an additional DNA quality and quantity check. 
The result interpretation can be affected by some issues. First of all, it should be taken into account that reference ranges change with gender and age, determining marked differences between children and adults. Furthermore, in case of certain immune deficiencies or autoimmune syndromes, a homeostatic peripheral replication of existing memory cells can be present, which dilutes TREC/KREC content and lowers the result of the assay when expressed only per 106 lymphocytes. This may lead to the erroneous conclusion that the thymic/bone marrow output is lower than it actually is. Therefore, it is advisable to always estimate lymphocyte neoproduction by also calculating TRECs/KRECs per ml of blood, which makes results independent of the proliferation rate of memory cells. Third, whatever is the unit of measure, the test result may lead to biased overestimates, due to persistence of some long-lived na&#239;ve lymphocytes that still bear TRECs or KRECs. Therefore, those who are interested in the best possible estimate of &#8220;true&#8221; lymphocyte neoproduction can take advantage of some more complicated mathematical models that also takes into account the lymphocyte apoptosis rate.15
An improvement of assay sensitivity to enhance its use is to perform TRECs and KRECs quantification starting from a limited amount of capillary whole blood spotted on Guthrie card filter paper. This modified version of the assay can be applied with success to the newborn screening of congenital immune deficiencies affecting lymphocyte production,12 and was proved beneficial in terms of cost-efficacy. This simplified version of assay also paves the way to new potential applications, especially considering the high number of potent immune modulating drugs that have been developed in the last few years. However, the &#8220;classical&#8221; TREC/KREC assay herein described has been already applied with success as a routine test in the follow up of children with severe congenital immune deficiencies following hematopoietic stem cell transplantation,4 or for the monitoring of the therapy outcome of autoimmune diseases and acquired immune deficiencies.6-9,11

T-hücresi reseptör eksizyon daire (TREC) ve K-silme rekombinasyon eksizyon daire (KRECs), genomik bir DNA yeniden birleştirme işlemi sırasında T ve sırasıyla A, B-hücreleri, bir oranda kesilmiş küçük daireselleşmiş DNA elemanları vardır giden Bir çok çeşitlendirilmiş T- repertuarına ve B-hücre reseptörleri oluşumu. Onlar hiçbir işlevi var, ama onlar kararlı ve taklit edilemeyen, çünkü onlar, böylece sadece iki kızı hücrelerin birinde ısrar, her hücre bölünmesinde sonra sulandırılır. Bu nedenle, periferik kandaki düzeylerinin timus, kemik iliği çıkışı bir tahmini olarak kabul edilebilir. TREC tahlil ölçüde timik çıktı kapsamını değerlendirmek için son 15 yılda kullanılmış olsa da, başlangıçta geliştirilen 1 Krec deneyi, B-hücresi çoğalmasını ve sağlık ve hastalık B-hücresi homeostasisin katkılarını, 2 ölçmek için ancak son zamanlarda kemik m marker olarak önerilmiştirçıktı ok. 3,4 Burada, biz TREC ve KRECs hem eşzamanlı ölçümü için geliştirilen yöntem açıklanmaktadır. 4 Bu kombine yöntem ile, gerçek zamanlı PCR ile DNA miktar ile ilişkili değişkenlik TREC, KRECs ve T-hücresi reseptörü alfa sabit fragmanlarını ihtiva eden bir üç-uç plazmid seyreltilmesi ile elde edilen eşsiz bir standart eğri kullanımı (TCRAC) ile ortadan kaldırılır 1: 1 oranında bir 1 geni. Bu TREC ve Krec kopya sayısında daha doğru bir değerlendirme sağlar. Ayrıca, aynı reaksiyonda iki hedef aynı anda miktar reaktif maliyet azalmasını sağlar. Önerilen TREC / Krec tahlil Şiddetli kombine immün yetmezlik (SCID), 4 Ortak Değişken İmmün Yetmezlik, 5 otoimmün hastalıklar, 6-8 ve HIV enfeksiyonu olan çocuklarda veya yetişkinlerde T- kapsamını ve B-hücresi neo-üretim ölçmek için yararlı olabilir . 9 Ayrıca, için kullanılabilirHastalar Krec kantifikasyonunu kullanılarak tespit edilebilir agamaglobülinemı SCID hastalarda altta yatan genetik bozukluklara rağmen TREC testi kullanılarak tanınan, çünkü Sonunda. 6-8 hematopoetik kök hücre nakli, 10 enzim replasmanı, 11 ve antiviral 9 veya bağışıklık tedaviler sonrasında bağışıklık ayilmanizi, ve , TREC / Krec deneyi de doğan tarama programlarına immün yetersizlikler tespit etmek için de kullanılabilir. Bu durumda, 12, test kan lekelenir küçük noktalar ekstre DNA üzerinde yapıldığında ve filtre kağıdı üzerinde kurutuldu gerekir, son derece hassas ve spesifik olmalıdır Hedef hastalıklar yanı sıra, oldukça üretilebilir ve maliyet-etkin bir. Wisconsin introduc ilk olunca test Krec miktar giriş rutin 2008 yılından bu yana ABD (WI, MA, CA) bazı bölgelerinde yapılmıştır immün için yenidoğan tarama, performanslarını artırmak gerekironun doğum tarama programında TREC e analizi. 13

<table border="0" cellpadding="0" cellspacing="0" width="861">
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			<td height="21" style="height:21px;width:296px;">Name of Reagent/ Equipment</td>
			<td style="width:211px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:235px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Histopaque-1077</td>
			<td style="width:211px;">Sigma Aldrich SRL</td>
			<td style="width:119px;">10771-500 ML</td>
			<td style="width:235px;">density gradient separation method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAamp DNA Blood Mini Kit (250)</td>
			<td style="width:211px;">QIAGEN</td>
			<td style="width:119px;">51106</td>
			<td style="width:235px;">DNA extraction</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;">Unmodified DNA Oligonucleotides HPSF 0.01 mmol</td>
			<td style="width:211px;">Eurofins MWG Operon/Carlo Erba Reagents S.r.l&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:235px;">Resuspend the lyophilized product to 100 picmol/&#181;l</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:296px;">AmpliTaq DNA Polymerase: including 10x Buffer II and 25mM MgCl2</td>
			<td style="width:211px;">Applied Biosystems/Life-Technologies</td>
			<td style="width:119px;">N8080156</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">GeneAmp dNTP Blend (100 mM)</td>
			<td style="width:211px;">Applied Biosystems/Life-Technologies</td>
			<td style="width:119px;">N8080261</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TOPO TA Cloning Kit for Subcloning</td>
			<td style="width:211px;">Invitrogen/Life-Technologies</td>
			<td>K4500-01</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XL1-Blue Subcloning Grade Competent Cells</td>
			<td style="width:211px;">Stratagene</td>
			<td>200130</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PureYield Plasmid Miniprep System</td>
			<td style="width:211px;">Promega</td>
			<td>A1223</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">SpeI 500U</td>
			<td style="width:211px;">New England Biolabs</td>
			<td>R0133S</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HindIII-HF 10,000 U</td>
			<td style="width:211px;">New England Biolabs</td>
			<td>R3104S</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PureYield Plasmid Midiprep System</td>
			<td style="width:211px;">Promega</td>
			<td>A2492</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">XhoI 5,000 U</td>
			<td style="width:211px;">New England Biolabs</td>
			<td>R0146S</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIS Utrapure</td>
			<td style="width:211px;">Sigma Aldrich SRL</td>
			<td>T1503</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td style="width:211px;">Sigma Aldrich SRL</td>
			<td>E5134</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TE buffer (1 mM TRIS and 0.1 mM EDTA)</td>
			<td style="width:211px;"></td>
			<td style="width:119px;"></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">TaqMan Universal PCR Master Mix</td>
			<td style="width:211px;">Applied Biosystems/Life-Technologies</td>
			<td style="width:119px;">4364338</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Dual labeled probes HPLC 0.01 mmol</td>
			<td style="width:211px;">Eurofins MWG Operon/Carlo Erba Reagents S.r.l&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:235px;">Resuspend the lyophilized product to 100 picmol/&#181;l</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoDrop 2000c spectrophotometer</td>
			<td style="width:211px;">ThermoFisher</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Applied Biosystems 2720 Thermal Cycler</td>
			<td style="width:211px;">Applied Biosystems/Life-Technologies</td>
			<td>4359659</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Fast 7500 Real-Time PCR system</td>
			<td style="width:211px;">Applied Biosystems/Life-Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">SDS Sequence Detection Software 1.4</td>
			<td style="width:211px;">Applied Biosystems/Life-Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:235px;"></td>
		</tr>
	</tbody>
</table>

simultaneous quantification of t-cell receptor excision circles (trecs) and k-deleting recombination excision circles (krecs) by real-time pcr

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106 cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.

Tahlil 87 sağlıklı kontrol temsili örneklemde gerçekleştirilmiştir: 42 çocuk 0 yaş - 17 (erkek / kadın: 25/17) ve 24 yaş 45 yetişkin - 60 (erkek / kadın: 29/16). Sonuçlar hesaplandı TREC 10 6 PBMC başına KRECs ve daha sonra kan ml&#39;si başına TREC ve KRECs olarak elde edildi. - 4 yıl TREC sayısı nedeniyle 0-3 çok keskin bir şekilde, özellikle de timus involüsyon, 4 yaşla birlikte azalmaktadır. Kadınlarda erkeklere göre daha hızla azalır, çünkü erişkinlerde, TREC sayısı da cinsiyet bağlıdır. 5 Bu, yaş ve cinsiyet muhtemelen temelinde farklılaştırılmış bir çok hassas eğer gereken uygun referans aralıkları, ayar sorunlar yaratabilir normallik tahmini gereklidir. <img alt="Şekil 3," src="/files/ftp_upload/52184/52184fig3display.jpg" /> Sağlıklı do Şekil 3. TREC miktar<html> NOR. TREC miktarı TREC / 10 sağlıklı çocuklar (A) ve aynı konularda (C, D) &#39;de TREC / ml olarak hesaplanmıştır sonra yetişkin (B) ve 6 hücreler tespit edilmiştir. Temizle daireler: kadın; dolu daireler: erkek. Çizgiler log dönüştürülmüş veriler kullanılarak lineer regresyon analizi ile elde edilmiştir. Çocuklarda, iki regresyon çizgileri daha iyi yaş TREC azalma değişken bir oranı temsil yerleştirilmiştir. Yetişkinlerde, kesik çizgiler erkeklerde (sürekli çizgi) daha yavaş kadınlarda görülen TREC, yaşla birlikte azalma eğilimi gösterir. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52184/52184fig3highres.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> Yetişkinlerde kemik iliği çıkış ömrü boyunca oldukça kararlı olan ve cinsiyet bağlı değildir, oysa KRECs sayısı, sadece çocuklarda TREC benzer bir desene sahip yaşla birlikte azalmaktadır. Her zaman &quot;&gt;:&quot; tutmak-together.within-sayfa = fo &quot;_content <img alt="Şekil 4," src="/files/ftp_upload/52184/52184fig4display.jpg" /> Şekil 4:. Sağlıklı donorlarda Krec miktar KRECs miktarı sağlıklı çocuklar için (A) ve aynı konularda (C, D) &#39;de KRECs / ml olarak hesaplanmıştır sonra yetişkin (B) ve KRECs / 10 6 hücre olarak belirlenmiştir. Temizle daireler: kadın; dolu daireler: erkek. Çizgiler log dönüştürülmüş veriler kullanılarak lineer regresyon analizi ile elde edilmiştir. Çocuklarda, iki regresyon çizgileri daha iyi yaşla birlikte azalma Krec değişken oranı temsil etmek takıldı. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52184/52184fig2highres.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> Temsili amaçlar için, basit log-lineer regresyon modelleri yaş TREC / Krec azalma desen tasvir etmek monte edildi, ama daha rafine doğrusal olmayan modeller i monte edilebilirn bir girişim daha yaşla birlikte görünüşte üstel Krec / TREC azalma tahmin etmek.

Watch this Scientific Journal Video about Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR at JoVE.com

Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

T-hücre reseptörleri Eksizyon Circles Eşzamanlı Niceleme (TREC) ve K-silme Rekombinasyon Eksizyon Circles (KRECs) Gerçek zamanlı PCR ile

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination ...

simultaneous-quantification-t-cell-receptor-excision-circles-trecs-k

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

16.4K Görüntüleme.  Spedali Civili di Brescia. Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

Video: Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Karşı Önceden tanımlanmış T Hücre Reseptör özgüllükleri Transnuclear Fare Toxoplasma gondii SCNT ile Elde Edilen

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

İmmünoloji ve Enfeksiyon

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

Gerçek zamanlı, TCR Eşzamanlı Görüntüleme ve İlişkili Sinyal Proteinler için TIRF Mikroskopi Tekniği

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Normal MSS Real-time PCR ve Nöro sırasında mikroglia içinde microRNA tespiti

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

T hücre gerçek zamanlı Canlı Görüntüleme Kompleks Oluşum Sinyal

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Miyeloid hücrelerin Dynamics Gerçek zamanlı Görüntüleme<em&gt; Süm<sup&gt; Min / +</sup</em&gt; Bağırsak Tümörleri Disk Eşodaklı Mikroskopi Spinning tarafından

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Yüksek verim Kantitatif Gerçek zamanlı RT-PCR Testi Türleri I ve III İnterferon Alttipleriyle İfadesi Profilleri belirlenmesi için

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

Kemik iliği projenitör hücrelerinin retroviral İletimi T-hücre reseptör Retrogenic fareler üretmek için

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

bir Genotiplendirme<em&gt; Staphylococcus aureus</em&gt; Tarafından Ribozomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

Gerçek zamanlı görüntüleme ve miktar iki aşamalı dolaşım akış sistemi kullanarak mantar biyofilm gelişimi

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

T-hücre reseptör üzerinden tarama kimyasal inhibitörü kütüphanelerin sinyal arabulucu tanımlaması

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...