Royal Prince Alfred ve Concord ÇHS ve normal bağırsak mukozasında taze örnekleri toplamak için ülkesine geri gönderilme Hastaneler Anatomik Patoloji Laboratuvarları personel teşekkür ederim. Iş Kanser Enstitüsü Yeni Güney Galler Translasyonel Programı Hibe tarafından finanse edildi.

<img src="/files/ftp_upload/3322/3322fig1.jpg" alt="Şekil 1" /> Şekil 1: CRC cerrahi örnek canlı hücreleri süspansiyon hazırlamak için İş akışı. 1. Klinik örnek ayrıştırılmasıyla Tüm numuneler X08-164 No&#39;lu Protokol&#39;ün altında bilgilendirilmiş onam ile Royal Prince Alfred Hastanesi (Camperdown, NSW, Avustralya) ve Concord Repatriation Hastanesi (Concord Batı, NSW, Avustralya) toplanmıştır. <ol><li> Kolorektal kanser (CRC), taze veya adenom numuneler ve normal bağırsak mukozasında tümörün en az 10 cm toplayın. 4 Hank dengeli tuz solüsyonu pH 7,3 (HBSS) ° C kadar rezeksiyon sonrası 12 saat için saklayın örnekleri. </li><li> Biyolojik güvenlik kabini sınıf II, tüm klinik örneklerde süreci, insan patojenler için emniyet kurallarına uyunuz. Iki neşter bıçak kullanarak bir Petri kabındaki 2 mm küp numuneler parçalara ayır. </li><li> 37 az 60 dakika zaman zaman nazik karıştırma ayrı Eppendorf tüpleri tümör ve normal doku inkübe ° C,% 2 kollajenaz tip 4 (Worthington, Lakewood, New Jersey, ABD) ve 0.1 (v / v) içeren RPMI 1640 orta eşit hacimde sığır pankreas% (w / v) deoksiribonükleaz (DNAz, Sigma-Aldrich). </li><li> Force 10 ml şırınga pistonu kullanarak, bir ince tel örgü süzgeç ile doku yarı sindirilmiş HBSS ile yıkama hücreleri yoluyla. </li><li> Hücre agrega kaldırmak için 200 mikron ve 50 mikron Filcon filtreler (BD Biosciences) ile hücre süspansiyonu sonucu iletin. DNA, mukus ve hücre agrega çoğu sızmalarından bu dizi kaldırılır. </li><li> Hücre süspansiyonları, 5 dakika boyunca 20 ° 400 xg&#39;de santrifüjleyin. </li><li> % 10 dimetil sulphoxide (DMSO) içeren ısı ile inaktive edilmiş FCS yeniden süspanse hücre pelletleri, -80 ° cryovials ve mağaza yavaş dondurma. Dondurma işlemi örnek mukus azaltmak eğilimindedir ve kırmızı kan hücreleri lyses. </li></ol> 2. Hücre yakalamak için örnek hazırlama <ol><li> 37 ° 10 ml DMSO yıkayın HBSS bir su banyosu ve tekrar süspansiyon hücreleri hızla örnekleri çözülme. </li><li> 5 dakika boyunca 20 ° 410 xg&#39;de hücre süspansiyonları santrifüjleyin. </li><li> Süpernatantı Durusu ve HBSS 500 mcL hücre pelletini tekrar süspansiyon haline getirin. </li><li> % 0,1 (w / v) oda sıcaklığında 20 dakika DNAz I ile örnek davranın. </li><li> Tripan mavi eşit hacmi ile her bir hücre süspansiyonu 10 mcL karıştırın ve karışımı 10 mcL hemasitometre içine yüklemek. 100 kat büyütme bir ışık mikroskobu kullanarak ölü hücrelerden boya alırken, tripan mavi dışlanma nedeniyle net görünen canlı hücreleri, saymak. En az 4 x 10 6 canlı hücreler mikroarray hücre yakalamak için gereklidir. </li><li> DNAz tedaviden sonra, 5 dakika boyunca 20 ° 410 xg&#39;de 10 ml HBSS ve santrifüj hücre süspansiyonu tekrar süspansiyon haline getirin. </li><li> RPMI 1640 süpernatantlar ve tekrar süspansiyon hücre pelletleri son hacim 200 mcL Durusu. </li></ol> 3. Antikor mikroarray hücre yakalama <ol><li> DotScan antikor mikroarray nitroselüloz bölümünde fosfat içine batırarak ıslatın yaklaşık 20 s için tamponlu salin (PBS) Dikkatle nitroselüloz bölümünde dokunmaktan kaçınarak, katlanmış Kimwipes mikroarray cam kenarları silin. </li><li> Nemli bir oda sağlamak için mikroarray inkübasyon tepsisine su ekleyin. Mikroarray odasına yerleştirin ve nemli nitroselüloz bölümünde üzerine RPMI 1640 yılında hücre süspansiyonu pipetle. Pipet bile hücrelerinin yayılmasını sağlamak için nitroselüloz her köşesinde düşer. </li><li> 1 saat boyunca 37 ° C&#39;de inkübe mikroarray&#39;ler Kuluçka hücrelerinin mikroarray antikorlar ile temas yerleşmek ve gelecek sağlar. Arazi antikorlar karşılık gelen yüzey antijenlerini eksprese eden hücrelerin Çekilecek. </li><li> Inkübasyondan sonra, bağlanmamış hücreleri (yıkama başına 20 sn) yıkayın için en az 15 ml PBS içeren üç olukları içine yavaşça ve dikey mikroarray&#39;ler daldırma. </li><li> Çapraz bağlayarak hücreleri ve antikor düzeltmek için% 3.7 (w / v) PBS içinde formaldehit hazırlayın. Yavaşça mikroarray nitroselüloz bölümü kapsayan yaklaşık 1 ml pipetle. Oda sıcaklığında 20 dakika inkübe edin. </li><li> Sonraki daldırma mikroarray&#39;ler PBS (15 ml, her biri) 30 s 3 değişiklik içine fazla formaldehit yıkayın. </li><li> Kenarlar ve arka cam slayt Kimwipes ile silin ve mikroarray DotScan tarayıcı süre kullanarak tarama, nitroselüloz bölümünde nemli. Optik tarama, karışık bir hücre popülasyonu örneğin CRC hücreleri, lökositler ve diğer stromal hücreler tümör antijeni ifade desen sağlar. </li></ol> 4. Çoğullama Floresan <ol><li> Mikroarray tarayıcıdan çıkarın ve engelleme tampon (% 2 w / v BSA ısı ile inaktive edilmiş insan AB serum, PBS, pH 7.3,% 2) 200 mcL uygulayın. Mikroarray tepsi 20 dakika oda sıcaklığında inkübe edin. </li><li> Multip hazırlayın(Biolegend, Eppendorf tüp, 10 mcL Alexa Fluor 647-anti-EpCAM, 20 mcL Fikoeritrin-anti-CD3 (1/7.5 son seyreltme Beckman Coulter, Gladesville, NSW, Avustralya, # IM12824): alüminyum folyo ile kaplanmış bir çözüm lexing San Diego, CA, ABD; 1 / 15 dilüsyon), ısı ile inaktive edilmiş insan AB serumu (Sigma-Aldrich, Castle Hill, NSW, Avustralya) ve engelleme tampon 118 mcL 2 mcL. </li><li> Mikroarray fazla engelleme tampon boşaltın ve düzgün yayılan nitroselüloz bölüm üzerine çoğullama çözüm pipetle. Oda sıcaklığında karanlıkta 30 dakika inkübe edin. </li><li> 15 ml taze PBS üç olukları içine dikey mikroarray batırın (her biri 30 sn). </li><li> Karanlık ve mağaza mikroarray&#39;ler kurumasını bekleyin, 4 ° C slayt kutusunda. Mikroarray floresan kaybı olmadan, 3 ay kadar karanlık saklanabilir. </li><li> Typhoon FLA 9000 - 50 çözünürlüğe sahip bir tarayıcı (GE Healthcare, Rydalmere, NSW, Avustralya) kullanılarak kuru mikroarray Tarama (532 nm lazer, PE 580 BP30 emisyon filtresi. Alexa 647 633 nm lazer ve 670 BP30 emisyon filtresi). Mikroarray&#39;ler nitroselüloz tarafı cam tarayıcı tepsi üzerindeki aşağı bakacak şekilde taranır. </li><li> TIFF dosyaları olarak floresan görüntü ve Photoshop seti kullanarak görüntü boyutu kaydet 17 x 25 cm ve çözünürlüğü 72 piksel / cm. Nokta yoğunluğu ile analiz etmek, DotScan analiz yazılımı görüntü alma. </li><li> DotReader nokta bağlayıcı deseni dijital bir görüntüsünü yakalar ve bir 8 bit grilik skalası (1-256 arası U) her bir antikor nokta hücre bağlanma yoğunluğu rakamlarla. Ara sıra non-spesifik izotip kontrol bağlanma ilgili immünglobulin isotypes antikorları için bağlayıcı değerler çıkarılır. % 100 yoğunlukta parlak nokta karşı her mikroarray Nokta floresan yoğunlukları normalize edildi. Sinyal / spot gücü bir xml dosyası (ham veri) ya da bir çubuk grafik olarak temsil edildi. Pdf dosyası (son rapor) </li><li> Mikroarray heatsmaps ve hiyerarşik kümeleme TM4 Mikroarray Software Suite (MultiExperiment Viewer (MeV) sürüm 4.4 kullanılarak yapılmıştır <a href="http://www.tm4.org/mev.html">http://www.tm4.org/mev.html</a> ). Hiyerarşik kümeleme tam bağlantı analizi ile MeV kullanarak arka plan arındırılmış veriler yapıldı. Öklid mesafe benzerlik ölçmek için kullanılır oldu. Eşit varyans ile 2-kuyruklu Student t-test sonuçlarının istatistiksel önemini belirlemek için kullanılmıştır. </li></ol> 5. Temsilcisi Sonuçlar: DotScan mikroarray Sonuçlar yinelenen dizileri arasında tutarlı hücre bağlayıcı desen göstermelidir. Güçlü hizalama nokta bağlama (CD44/CD29) dizi alana yerleştirilmiş olmak için bir kılavuz sağlar. Şekil 2, optimum hücre yakalama ve çoğullama bir örnek gösterir. Şekil 3 hücre yakalama ve olası çözümleri sırasında karşılaşılan bazı ortak sorunları gösterir. Mikroarray hücre bağlayıcı sonuçlar 1 ile 256 arasında değişen bir grilik ölçekte ifade nokta yoğunlukları ölçerek belirlenebilir. Şekil 4 hiyerarşik kümeleme ile İlgi haritası EpCAM-Alexa 647 antikor ile boyandı 58 cerrahi CRC örneklerinden sayısal veriler, gösterir. Numune sayısı sınırlı olsa da, aynı sahnede CRC aynı grupta küme eğilimindedir. <img src="/files/ftp_upload/3322/3322fig2.jpg" alt="Şekil 2" /> Şekil 2 klinik kolorektal kanser tümörü (Avustralya Klinik-Patolojik Evreleme, ACP evre B1) Hücre bağlayıcı desen. Yinelenen mikroarray (özetlendiği gibi) sol yarısı için antikor yerlerini gösteren (a) DotScan antikor anahtarı. Üst bölümde DotScan lösemi mikroarray orijinal 82 antikorlar içerir. Spesifik yüzey antijenleri ilgili ek bir 40 antikorlar, bir CRC &#39;uydu&#39; mikroarray olarak eklendi, literatürde up-regüle bulundu. Alt kısmında izotip kontrol antikorlar mikroarray bağlayıcı CRC hücrelerinin (b) Optik görüntü oluşur. T-hücrelerinin (c) CD3 floresan görüntü. (D) CRC hücreleri gösteren EpCAM floresan görüntü. <img src="/files/ftp_upload/3322/3322fig3.jpg" alt="Şekil 3" /> Şekil 3, kötü DotScan sonuçları ve olası çözümleri örnekleri . (A) Düşük hücre bağlayıcı çözüm:; mikroarray üzerinde kuluçka önce en aza indirmek için örnek ısı ile inaktive edilmiş insan AB serum eklemek çözüm en azından 4x106 canlı hücreler (b) bağlayıcı İzotip kontrolü bağlayıcı ve non-spesifik bir hücre dizisi üzerinde emin olun. izotip kontrol bağlayıcı. Bazen, nitroselüloz hücreleri non-spesifik bağlayıcı bir miktar CRC örnekleri ile ortaya çıkar ve sonuçları önemli ölçüde etkilemez. (C) Selülozik inkübasyon sırasında kuruma; çözüm: örnek tüm nitroselüloz bölümü kapsar ve mikroarray düz bir yüzey üzerinde inkübe sağlamak. (D) Yüksek arka plan eserler; çözüm: sağlamakmikroarray aşağıdaki inkübasyon iyice yıkanır. <img src="/files/ftp_upload/3322/3322fig4.jpg" alt="Şekil 4" /> Şekil 4 DotScan analiz yazılımı, 1 ile 256 arasında değişen bir grilik ölçekte bağlayıcı hücre yoğunlukları temsil eden çubuk grafikler oluşturulur. Eksen üzerinde Sayılar CD antijenleri bakın. Κ, λ, immünglobulin hafif zincirleri;; sIg, yüzey immünglobulin; DCC, kolorektal kanser protein silinir; EGFR, epidermal büyüme faktörü reseptörü; FAP, fibroblast aktivasyonu protein, HLA-A, B, Diğer kısaltmalar TCR, T-hücre reseptör A, B, C, sırasıyla C HLA-DR, insan lökosit antijenleri DR ve; MİKA, MHC sınıf I zincir-ilişkili protein A, MMP-14, matris metallopeptidase 14; PIGR, polimerik immünglobulin reseptör; TSP-1, trombospondin-1; Mabthera, insanlara anti-CD20 <a href="http://www.jove.com/files/ftp_upload/3322/3322fig4large.jpg">büyük resim görmek için buraya tıklayın</a> .

<ol>
	<li>Steinert, R., Buschmann, T., vander Linden, M., Fels, L. M., Lippert, H., Reymond, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+proteomics+in+the+diagnosis+and+outcome+prediction+in+colorectal+cancer.">The role of proteomics in the diagnosis and outcome prediction in colorectal cancer.</a> Technol. Cancer. Res. Treat. 1, 297 (2002).</li><li>Eifel, P., Axelson, J. A., Costa, J., Crowley, J., Curran, W. J., Deshler, A., Fulton, S., Hendricks, C. B., Kemeny, M., Kornblith, A. B., Louis, T. A., Markman, M., Mayer, R., Roter, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National+Institutes+of+Health+Consensus+Development+Conference+Statement:+adjuvant+therapy+for+breast+cancer.">National Institutes of Health Consensus Development Conference Statement: adjuvant therapy for breast cancer.</a> J. Natl. Canc. Inst. 93, 979 (2001).</li><li>Swerdlow, S. H., Campo, E., Harris, H. L., Jaffe, E. S., Pileri, S. A., Stein, H., Thiele, J., Vardiman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WHO+classification+of+tumour+of+haematopoietic+and+lymphoid+tissues.">WHO classification of tumour of haematopoietic and lymphoid tissues.</a> IARC WHO Classification of Tumours. 2, (2008).</li><li>Xiao, G. G., Recker, R. R., Deng, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+proteomics+and+cancer+biomarker+discovery.">Recent advances in proteomics and cancer biomarker discovery.</a> Clin. Med. Oncol. , (2008).</li><li>Belov, L., Mulligan, S. P., Barber, N., Woolfson, A., Scott, M., Stoner, K., Chrisp, J. S., Sewell, W. A., Bradstock, K. F., Bandall, L., Pascovici, D. S., Thomas, M., Erber, W., Huang, P., 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=Analysis+of+human+leukaemias+and+lymphomas+using+extensive+immunophenotypes+from+an+antibody+microarray.">Analysis of human leukaemias and lymphomas using extensive immunophenotypes from an antibody microarray.</a> Br. J. Haematol. 135, 184 (2006).</li><li>Belov, L., Huang, P., Barber, N., Mulligan, S. P., Christopherson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+repertories+of+surface+antigens+on+leukemias+using+an+antibody+microarray.">Identification of repertories of surface antigens on leukemias using an antibody microarray.</a> Proteomics. 3, 2147 (2003).</li><li>Zhou, J., Belov, L., Huang, P. Y., Shin, J., Solomon, M. J., Chapuis, P. H., Bokey, L., Chan, C., Clarke, C., Clarke, S. J., Christopherson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+antigen+profiling+of+colorectal+cancer+using+antibody+microarrays+with+fluorescence+multiplexing.">Surface antigen profiling of colorectal cancer using antibody microarrays with fluorescence multiplexing.</a> J. Immunol. Methods. 355 (1-2), 40-51 (2010).</li><li>Ellmark, P., Belov, L., Huang, P., Lee, C. S., Solomon, M. J., Morgan, D. K., Christopherson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+detection+of+surface+molecules+on+colorectal+cancers.">Multiplex detection of surface molecules on colorectal cancers.</a> Proteomics. 6, 1791 (2006).</li><li>Pearson, J. P., Allen, A., Hutton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheology+of+mucin.">Rheology of mucin.</a> Methods Mol. Biol. 125, 99 (2000).</li><li>Yang, Y. H., Dudoit, S., Luu, P., Lin, D. M., Peng, V., Ngai, J., Speed, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+for+cDNA+microarray+data:+a+robust+composite+method+addressing+single+and+multiple+slide+systematic+variation.">Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation.</a> Nucleic Acids Res. 30, 15 (2002).</li><li>Al Saeed, ., 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=TMA:+A+free,+open-source+system+for+microarray+data+management+and+analysis.">TMA: A free, open-source system for microarray data management and analysis.</a> BioTechniques. 34, 374-378 (2003).</li></ol>

Çıkar çatışması ilan etti.

Bu video, DotScan antikor mikroarray CRC doku hücre popülasyonlarının için yüzey antijeni profillerini incelemek için basit, yarı-kantitatif bir şekilde nasıl kullanılabileceğini göstermektedir. İnkübasyon sırasında bütün hücrelerin antikor nokta firma bağlanma için gerekli olan enerji bağımlı süreçler (örn., antijen kapak kapatma ve / veya pseudopodia oluşumu) görünür, çünkü doku yaşayabilir bir tek hücre süspansiyonu elde etme, deneyin başarısı içi...

The author's email has been corrected in the publication of <a href="http://www.jove.com/video/3322">Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing</a>. There was an error with the author, Jerry Zhou's, email. The author's email has been updated to:
j.zhou@uws.edu.au
from:
jzho7551@mail.usyd.edu.au

Erratum: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

<table border="1"><tbody><tr><th> Reaktif ya da ekipman Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> Hanks &#39;dengeli tuz solüsyonu </td><td> Sigma-Aldrich </td><td> H6136-10X1L </td><td> 25 mM HEPES (Sigma # H3375) ile tamponlanmış </td></tr><tr><td> Airpure biyolojik güvenlik kabini sınıf II </td><td> Westinghouse </td><td> 1687-2340/612 </td><td></td></tr><tr><td> Cerrahi bıçaklar </td><td> Livingstone </td><td> 090609 </td><td> 100 adetlik ambalaj </td></tr><tr><td> 2 mM HEPES RPMI 1640 </td><td> Sigma-Aldrich </td><td> R4130-10X1L </td><td></td></tr><tr><td> Kollajenaz tip 4 </td><td> Worthington </td><td> 4188 </td><td></td></tr><tr><td> Deoksiribonükleaz 1 </td><td> Sigma-Aldrich </td><td> DN25-1G </td><td></td></tr><tr><td> Terumo Şırınga (10 mL) </td><td> Terumo </td><td> SS 10 L </td><td> 100 Kutu </td></tr><tr><td> Filcon filtre (200 mikron) </td><td> BD Biosciences </td><td> 340615 </td><td></td></tr><tr><td> Filcon filtresi (50 mikron) </td><td> Filcon filtresi (50 mikron) </td><td> Filcon filtresi (50 mikron) Filcon filtresi (50 mikron) 340.603 </td><td></td></tr><tr><td> Fetal buzağı serumu </td><td> Gibco / Invitrogen </td><td> 10099-141 </td><td></td></tr><tr><td> 5810 R Santrifüj </td><td> Eppendorf </td><td> 7017 </td><td></td></tr><tr><td> Dimetil sulphoxide </td><td> Sigma-Aldrich </td><td> D2650 </td><td></td></tr><tr><td> Tripan mavi </td><td> Sigma-Aldrich </td><td> T8154 </td><td></td></tr><tr><td> Hemocymeter Technocolor Neubar </td><td> Hirschmann </td><td> mevcut değil </td><td></td></tr><tr><td> Işık mikroskobu </td><td> Nikon </td><td> Nikon TMS </td><td></td></tr><tr><td> Cyrovial tüpler </td><td> Greiner biyo-bir </td><td> 121278 </td><td></td></tr><tr><td> Cryo dondurma contrainer </td><td> Nalgene </td><td> 5100-0001 </td><td></td></tr><tr><td> DotScan antikor mikroarray kiti </td><td> Medsaic </td><td> mevcut değil </td><td></td></tr><tr><td> DotScan mikroarray yıkama tepsisi </td><td> Medsaic </td><td> mevcut değil </td><td></td></tr><tr><td> Kimwipes </td><td> Kimberly-Clark </td><td> 4103 </td><td></td></tr><tr><td> 37% Formaldehit </td><td> Sigma-Aldrich </td><td> F1635-500ML </td><td></td></tr><tr><td> DotReaderTM </td><td> Medsaic </td><td> mevcut değil </td><td></td></tr><tr><td> Sığır serum albumini </td><td> Sigma-Aldrich </td><td> A9418-10G </td><td></td></tr><tr><td> Isı ile inaktive AB serum% 2 </td><td> Invitrogen </td><td> 34005100 </td><td></td></tr><tr><td> Fikoeritrin-konjuge CD3 </td><td> Beckman Coulter </td><td> ET386 </td><td></td></tr><tr><td> AlexaFluor647-konjuge EpCAM </td><td> BioLegend </td><td> 324212 </td><td></td></tr><tr><td> Typhoon FLA 9000 </td><td> GE Healthcare </td><td> 28-9558-08 </td><td> 532 nm lazer, PE 580 BP30 emisyon filtresi. 633 nm lazer ve BP30 Alexa647 için 670 emisyon filtresi </td></tr><tr><td> MultiExperiment Görüntüleyici v4.4 </td><td> TM4 Mikroarray Software Suite </td><td> Open - kaynak yazılım (Ref 11) </td><td></td></tr></tbody></table>

colorectal cancer cell surface protein profiling using an antibody microarray and fluorescence multiplexing

ÇHS geçerli prognoz ve sınıflandırma sistemleri evreleme histopatolojik ve klinik bulgular entegre dayanır. Ancak, ÇHS vakaların çoğunda protein ekspresyonu ve post-translasyonel değişiklik 1 değişiklik sayısız mutasyonlar, hücre disfonksiyonu sonucu. CRC potansiyel prognostik veya metastatik biyobelirteçleri farklılaşmasını (CD) antijenlerinin küme de dahil olmak üzere hücre yüzey antijenleri, bir dizi olarak tespit edilmiştir. Bu antijenler genellikle kendi ifadesi olarak tümör progresyonu veya diğer hücre türleri gibi ile etkileşim değişiklikleri ideal biyobelirteçleri yapmak tümör infiltre eden lenfositler (TILs) ve tümör ilişkili makrofajlar (TAM). Kanseri alt sınıflandırma ve prognoz tahmini için immünhistokimyasal (İHK) kullanımı bazı tümör tiplerinde 2,3 için kurulmuştur. Ancak, tek bir &#39;işaretleyici&#39; den büyük klinik-patolojik prognostik önemi gösterilen veya rutin patoloji raporlama tüm CRC durumlarda kullanılmak üzere geniş bir kabul görmüştür. Hastalık fenotipleri prognostik tabakalaşma bir daha yeni bir yaklaşım, birden çok &#39;işaretleyicileri kullanarak yüzey protein profilleri dayanmaktadır. ITRAQ olarak proteomik teknikler kullanarak tümörlerin ifade profil biomarkers4 keşfi için güçlü bir araç olmasına rağmen, teşhis laboratuvarlarında rutin kullanımı için en uygun değildir ve karışık bir nüfus farklı hücre tipleri ayırt edemez. Buna ek olarak, tümör dokusu büyük miktarda bu yöntemlerle saflaştırılmış plazma zarı glikoproteinler profil için gereklidir. Bu video DotScan CRC antikor mikroarray kullanarak ayrıştırılmış CRC örneklerinden canlı hücreler yüzey proteom profil için basit bir yöntem tanımlanmıştır. 122-antikor mikroarray KRK için 40 potansiyel prognostik belirteçlerin tespiti için bir uydu bölge ile birlikte, bir dizi özel soyundan lökosit belirteçler, adezyon moleküllerinin, reseptörleri ve enflamasyon ve immün yanıt 5 belirteçleri tanıyan standart bir 82-antikor bölgesi oluşur . Hücreler, yalnızca ilgili antijen ifade antikorlar yakalanır. Nokta başına optik tarama tarafından belirlenen hücre yoğunluğu, bu antijen, antijen ve antikor 6 yakınlık ifade düzeyi ifade hücrelerinin oranını yansıtır. CRC doku ya da normal bağırsak mukoza hücrelerinin karışık nüfusu, optik taramaları immünfenotip yansıtmaktadır. Floresan çoğullama sonra dizi yakalanan ilgi hücreleri seçilen alt popülasyonlarda profil kullanılabilir. Örneğin, Alexa 647 anti-epitel hücre adezyon molekülü (EpCAM CD326) Fikoeritrin-anti-CD3, kullanılmış iken, CRC hücreleri ve normal bağırsak mukozasında epitel hücreleri algılamak için kullanılan bir pan-epitelyal farklılaşma antijen T-hücrelerinin 7 infiltre algılamak için. DotScan CRC mikroarray anatomik tabanlı CRC evreleme sistemi için bir tanı alternatif prototip olmalıdır.

Watch this Scientific Journal Video about Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing at JoVE.com

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

Kolorektal kanser (CRC) uygulanabilir tek bir hücre üretmek için, daha sonra yüzey antijenleri (DotScan ÇHS mikroarray) tanıyan özelleştirilmiş antikor mikrodizinlerinde yakalanır ayrıştırılmasıyla için bir prosedür nitelendirdi. Mikroarray bağlı hücrelerin alt popülasyonlarda floresan boya ile monoklonal antikorlar kullanılarak floresans çoklama profilli olabilir.

Kolorektal Kanser Hücre Yüzey Protein Profil Antikor Mikroarray ve Floresans Multiplexing

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

colorectal-cancer-cell-surface-protein-profiling-using-an-antibody

Research

JoVE Journal

Immunology and Infection

13.9K Görüntüleme.  University of Sydney. Kolorektal kanser (CRC) uygulanabilir tek bir hücre üretmek için, daha sonra yüzey antijenleri (DotScan ÇHS mikroarray) tanıyan özelleştirilmiş antikor mikrodizinlerinde yakalanır ayrıştırılmasıyla için bir prosedür nitelendirdi. Mikroarray bağlı hücrelerin alt popülasyonlarda floresan boya ile monoklonal antikorlar kullanılarak floresans çoklama profilli olabilir.

Video: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

Viral Patojenler Klinik Örnekler Ekranına bir Pan-Viral Mikroarray Testi (Virochip)

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

İmmünoloji ve Enfeksiyon

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular localization can provide insights into the functional role of bacterial proteins. Surface localization of bacterial proteins is a key step towards identification of virulence factors involved in mechanisms of pathogenicity. 
Methods for fractionating leptospiral membranes 2-5 may be selective for a certain class of outer-membrane proteins (OMPs), such as lipoproteins vs. transmembrane OMPs, and therefore lead to misclassification. This likely is due to structural differences and how they are associated to the outer membrane. Lipoproteins are associated with membranes via a hydrophobic interaction between the N-terminal lipid moiety (three fatty acids) and the lipid bilayer phospholipids 6, 7. In contrast, transmembrane OMPs are typically integrated into the lipid bilayer by amphipathic &beta;-sheets arranged in a barrel-like structure 8, 9. In addition, presence of a protein in the outer-membrane does not necessarily guarantee that the protein or its domains are exposed on the surface. Spirochetal outer membranes are known to be fragile and therefore necessitate methods involving gentle manipulation of cells and inclusion of sub-surface protein controls to assess the integrity of the outer membrane.
Here, we present an immunofluorescence assay (IFA) method to directly assess surface exposure of proteins on intact leptospires. This method is based on recognition of leptospiral surface proteins by antigen-specific antibodies. Herein, antibodies specific for OmpL5410 are detetcted aftero binding to native, surface exposed epitopes. Comparison of antibody reactivity to intact versus permeabilized cells enables evaluation of cellular distribution and whether or not a protein is selectively present on leptospiral surface. The integrity of outer membrane should be assessed using antibody to one or more subsurface proteins, preferably located in the periplasm.
The surface IFA method can be used to analyze surface exposure of any leptospiral protein to which specific antibodies are available. Both the usefulness and limitation of the method depends on whether the antibodies employed are able to bind to native epitopes. Since antibodies often are raised against recombinant proteins, epitopes of native, surface-exposed proteins may not be recognized. Nevertheless, the surface IFA method is a valuable tool for studying components of intact bacterial surfaces. This method can be applied not only for leptospires but also other spirochetes and gram-negative bacteria. For stronger conclusions regarding surface-exposure of OMPs, a comprehensive approach involving several cell localization methods is recommended 10.

An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction.

Yüzey maruz Leptospiral Proteinler immuno-floresans Testi

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Tek hücreli Analizi Bacillus subtilis</emFloresan Mikroskobu ve Akım Sitometri Kullanma> Biyofilmler

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Canlı Hücre Floresan Mikroskopi kullanma Bakteriyel Toksin İsteyerek Yanıtların Görselleştirme

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Ana-Patojen protein-protein etkileşimleri ve Peyzaj karakterize etmek Karşılaştırmalı Bir Yaklaşım

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Influenza A Virüsü hemaglutininler aviditesi ve özgünlüğünün değerlendirilmesi için Minyatür Glycan Mikroarray Testi

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, immunodiagnostics and more recently as therapeutic agents. Traditional methods for labeling antibodies with small molecules require purified antibodies at relatively high concentration, involve multiple dialysis steps and have limited throughput. However, several applications, including the field of Antibody Drug Conjugates (ADCs), will benefit from new methods that will allow labeling of antibodies directly from cell media. Such methods may allow antibodies to be screened in biologically relevant assays, for example, the receptor-mediated antibody internalization assay in the case of ADCs. Here, we describe a method (on-bead method) that enables labeling of small amounts of antibodies directly from cell media. This approach utilizes high capacity magnetic Protein A and Protein G affinity beads to capture antibodies from the cell media followed by labeling with small molecules using either amine or thiol chemistry and subsequent elution of the labeled antibodies. Taking fluorescent dyes as surrogates for small molecules, we demonstrate the on-bead labeling of three different mouse antibodies directly from cell media using both amine and thiol labeling chemistry. The high binding affinity of antibodies to Protein A and Protein G ensures high recoveries as well as high purity of the labeled antibodies. In addition, use of magnetic beads allows multiple samples to be handled manually, thereby significantly improving labeling throughput.

The on-bead method for labeling antibodies with small molecules enables labeling of a small amount of antibodies directly from cell media. This method is compatible with amine and thiol chemistry, and can handle multiple samples in parallel, manually or using automated platforms.

Floresan Boyalar ile Antikor Etiketleme Manyetik Protein A ve Protein G Boncuk kullanma

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

Bir Protein Mikroarray tahlil antijen spesifik antikor yanıt aşağıdaki Clostridium difficile enfeksiyon serolojik belirlenmesi için

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

Tek hücreli Nefelometri mRNA ve yüzey Protein Simian virüs, immün yetmezlik enfekte CD4 ifadede+ T hücreleri izole Rhesus makak üzerinden

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.