Vanessa Garcia is funded by a Queen Elizabeth II Ontario Graduate Scholarship in Science and Technology and Cory Batenchuk by a Natural Sciences and Engineering Research Council fellowship.

1. Numune Hazırlama <ol><li> 96-yuvalı plakalar içine titrasyon ve transferi için lusiferaz sentezleyen virüsün (bir örnek olarak VSVΔ51-Fluc) içeren numuneler elde edilir. Alternatif olarak, doğrudan, 96 oyuklu doku kültür plakaları ve kullanım yüzer enfeksiyonların gerçekleştirin. </li><li> Standart eğriler dahil işlenmemiş iki sütun bırakın. Daha sonraki bir tarihte -80 ° C ve titrede deney (40 saat post-enfeksiyon) veya mağaza sonunda 96-yuvalı plakalar, titresi doğrudan yapılan deneyler için. </li></ol> Virüs titrasyonu için, permisif ve Hücreler 2. Hazırlık <ol><li> 24 saat önce hassas olarak ölçülmesi için,% 10 fetal inek serumu ihtiva eden Dulbecco&#39;nun Modifiye edilmiş Eagle ortamında 2.5 x 10 5 hücre / ml (DMEM) (FBS), 30 mM HEPES konsantrasyonunda Vero hücrelerinin bir süspansiyon hazırlamak, ve% 1 penisilin / kültürlendi. NOT: Bu protokol Vero hücreleri kullanır rağmen, VSV enfeksiyon için uygun herhangi bir müsamahakar hücre hattı olabilir bde kullanılabilmektedir. </li><li> Bir mikro-dağıtıcı kullanılarak 96 oyuklu beyaz bir katı, düz tabanlı plakalar içerisinde 2.5 x 10 4 hücreleri (100 ul) Tohum. <ol><li> Plaka başına hücre süspansiyonu 12 ml artı astar boyunca 5 ml hazırlayın. </li><li> Steril 50 ml su ile yıkama, borunun temizleyin mikro dağıtıcı kaseti. </li><li> Hücre süspansiyonu ile mikroplaka dağıtıcı çizgileri doldurmak ve hücre 5 ml süspansiyon akışı sağlar. </li><li> Beyaz bir duvarlı 96-çukurlu bir levhanın her çukuruna 100 ul dağıtmak bir program seçin. Koyun, ve ek plakalar için gerektiği gibi tekrarlayınız. Opak dipli beyaz duvarlı plakalar hücre sağlığı ve yoğunluk doğrulanması için kullanıldığında aynı zamanda, bir 96-açık görüş düz tabanlı plaka içinde bir kuyu kaç tohum. </li><li> geri orijinal kabın içine bitmiş, floş hücreler. Borular yoluyla ılık steril 50 ml su, ardından% 70 etanol, 50 ml çalıştırarak kaseti temizleyin. </li><li> Emin kaset ve tüp ap olunpropriately kullanımları arasındaki temizlenmelidir. </li></ol></li><li> Nemlendirilmiş bir% 5 CO2 inkübatöründe 37 ° C&#39;de 24 saat boyunca inkübe hücreleri. </li></ol> Viral Standart Eğrisi 3. hazırlanması <ol><li> 10 8 pfu / ml, 10 7 pfu / ml serum içermeyen DMEM içinde VSVΔ51-Fluc bir standart eğri hazırlamak, aşağıdaki şekilde, Vero hücreleri üzerine transfer edildikten sonra, ml başına plak oluşturucu birim (pfu), nihai konsantrasyon olduğu 10 6 pfu / ml, 10 5 pfu / mi, 10 4 pfu / ml, 10 3 pfu / mi, 10 2 pfu / ml ve 10 1 pfu / ml olmuştur. Vero hücrelerinin plaka artı ekstra% 10 başına her bir konsantrasyonu 50 ul hazırlayın. Not: lusiferaz virüs stokunun titresi kesin miktar sağlayacak bir standart eğri hazırlamak için klasik bir şekilde 10 değerlendirilecek gerekir. Aksi takdirde, miktar ölçümü keyfi ayarlayarak, viral titresi kesin titre bilgi olmaksızın elde edilebilir göreseyreltme adımlar. Örneğin, standart eğrinin birinci seyreltme 10 8, viral birimleri ve 10 7 ve böylece için aşağıdaki 1/10 seyreltme için ayarlanmış olabilir. Bu bağlamda, kesin olmayacağı gibi kesin miktar önceden belirlenmiş bir örneğe göre daha kat değişiklik olarak ifade değerleri gerekmektedir. </li></ol> Permisif hücreler üzerine Örnek süpernatanlarından 4. Transferi <ol><li> Tekli tabakaları, en azından% 95 birleştiğinde olduğunu teyit etmek üzere, bir ışık mikroskobu altında berrak tabanlı 96 oyuklu bir plaka içerisinde kaplama Vero hücreleri kontrol ediniz. </li><li> Beyaz duvarlı plakalarda tohumlanmıştır Vero hücreleri üzerine örnek süpernatan 25 ul aktarın. Standart eğrileri için belirlenen 2 sütun halinde süpernatanlarm havale etmeyin. Not: Bu, bir kanal 96, sıvı işleyici kullanarak tek bir plaka üzerinde tüm oyuklara için eş zamanlı olarak yapılabilir. </li><li> 8 veya 12 kanallı bir çok kanallı pipet kullanılarak, in Vero hücrelerinde edilmesi için Adım 3&#39;te hazırlanan standart eğrinin her bir seyreltinin 25 ul2 belirlenen sütun. </li><li> Oda sıcaklığında 430 x g&#39;de 5 dakika boyunca santrifüje plakalar. </li><li> Nemlendirilmiş bir% 5 CO2 inkübatöründe 37 ° C&#39;de 5 saat boyunca inkübe edin. </li></ol> Hücre canlılığı 5. Değerlendirme <ol><li> Not: açık, 96 oyuklu plakalar içinde yapılan enfeksiyon deneylerden elde edilen yüzer başlayarak zaman, örnek canlılık gibi resazurin gibi bir hücre canlılığı işaret eden bir boya ile miktar tayini önce değerlendirilebilir. </li><li> Virüs ve hücreleri içeren örneklerin, 96 oyuklu plakanın her oyuğundaki hacminin% 10&#39;una eşit bir miktarda resazurin ekleyin. Cell-only sırasıyla% 100 ve% 0 canlılığı değerlerini belirlemek için medya sadece kontrol yanı sıra kontrol içerir. </li><li> 2-4 saat (hücre tipine ve boya ticari olarak temin edilebilir ya da yeniden oluşturulan toz konsantrasyonuna bağlı olarak değişir inkübasyon süresi) sonra, okuma ve bir floresan plaka okuyucu kullanarak (530-560 uyarma, 590 emisyon) kullanarak sinyali kaydedilir. Rapor hücre viabilix% 100 formülüne Nispi metabolik aktivite = - (- negatif kontrol sinyali) / (Hücre tek sinyali kontrol negatif kontrol sinyali) (Test Örnek sinyali) &#39;e uygun bir numune için ty </li></ol> Luciferase Yüzey 6. hazırlanması <ol><li> 30 dakika 5 saat inkübasyonun sonunda önce, steril fosfat tamponlu tuzlu su (PBS) içinde 2 mg / ml bir solüsyon elde etmek için, lusiferin hazırlar. Plaka başına 2.5 ml artı bir ilave 2 ml hazırlayın. Işıktan çözüm koruyun. Not: lusiferin da günün erken hazırlandı ve kullanılana kadar 4 ° C&#39;de muhafaza edilebilir. </li></ol> 7. Okuma Biyoparlaklık <ol><li> 5 saatlik bir bekletme süresinden sonra, beyaz bir katı plakalar içinde Vero hücreleri her çukuruna luciferase 25 ul ekle. Elle veya luminometreye entegre otomatik bir dağıtıcı ile lusiferin ekleyin. Not: sadece tek ve bitiş noktasına sahip bir aletin kullanılması, bir lusiferaz uyumlu bir parçalama tampon tamponundan eklenmesini gerektirebilir okurfer tutarlılığını artırmak için lusiferin tabakayı eklemeden önce. </li><li> Aşağıdaki parametrelerle plakaları okuyun: <ol><li> 5 saniye çalkalayın. </li><li> 30 saniye bekleyin. </li><li> Uygun bir sabit hassasiyet / poz değeriyle luminescence okuyun. Seçeneği enstrüman üzerinde varsa, çok noktalı doğruluğunu geliştirmek için (örneğin, 3 x 3 matrisi) okur kullanın. </li></ol></li><li> Kayıt ve sayısal biyolüminesens yoğunluğunu kaydedin. </li><li> Otomatik dağıtıcı eklemek için kullanılır ise lusiferin lusiferin ve başbakan ılık steril su ile hatları temizlemek. </li></ol> 8. Veri Analizi <ol><li> Doğru sonuçlar için, standart eğrileri bir Tepesi denklem oluşturmak için non-lineer regresyon çözmek. Viral ekspresyon birimden titre örnekleri için bu denklem uygulanır. Bazı virüsler veya durumlar, doğrusal bir ilişki ile standart eğri üretebilir; Bu durumda lineer bir denklem çözmek. </li></ol>

<ol>
	<li>Grigorov, B., Rabilloud, J., Lawrence, P., Gerlier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+titration+of+measles+and+other+viruses:+optimization+with+determination+of+replication+cycle+length.">Rapid titration of measles and other viruses: optimization with determination of replication cycle length.</a> PLoS One. 6 (e24135), (2011).</li><li>Lizee, G., 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=Real-time+quantitative+reverse+transcriptase-polymerase+chain+reaction+as+a+method+for+determining+lentiviral+vector+titers+and+measuring+transgene+expression.">Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression.</a> Hum Gene Ther. 14, 497-507 (2003).</li><li>McSharry, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uses+of+flow+cytometry+in+virology.">Uses of flow cytometry in virology.</a> Clin Microbiol Rev. 7, 576-604 (1994).</li><li>Watcharatanyatip, K., 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=Multispecies+detection+of+antibodies+to+influenza+A+viruses+by+a+double-antigen+sandwich+ELISA.">Multispecies detection of antibodies to influenza A viruses by a double-antigen sandwich ELISA.</a> J Virol Methods. 163, 238-243 (2010).</li><li>Dawson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+Direct+Quantification+of+Viruses+in+Solution+Using+the+ViroCyt+Virus+Counter.">Rapid, Direct Quantification of Viruses in Solution Using the ViroCyt Virus Counter.</a> Journal of Biomolecular Techniques. 23 (S10), (2012).</li><li>Snyder, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV+and+RT-PCR:+true+or+false.">AAV and RT-PCR: true or false.</a> Mol Ther. 1, 389-390 (2000).</li><li>Diallo, J. S., Roy, D., Abdelbary, H., De Silva, N., Bell, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+infection+of+live+tissue+with+oncolytic+viruses.">Ex vivo infection of live tissue with oncolytic viruses.</a> J Vis Exp. 52, (2011).</li><li>Gaush, C. R., Smith, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+and+plaque+assay+of+influenza+virus+in+an+established+line+of+canine+kidney+cells.">Replication and plaque assay of influenza virus in an established line of canine kidney cells.</a> Appl Microbiol. 16, 588-594 (1968).</li><li>Green, M., Loewenstein, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adenoviruses:+propagation,+purification,+quantification,+and+storage.">Human adenoviruses: propagation, purification, quantification, and storage.</a> Curr Protoc Microbiol. 14 (14C 11), (2006).</li><li>Diallo, J. S., Vaha-Koskela, M., Le Boeuf, F., Bell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation,+purification,+and+in+vivo+testing+of+oncolytic+vesicular+stomatitis+virus+strains.">Propagation, purification, and in vivo testing of oncolytic vesicular stomatitis virus strains.</a> Methods Mol Biol. 797, 127-140 (2012).</li><li>Hoenen, T., Groseth, A., Callison, J., Takada, A., Feldmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+Ebola+virus+expressing+luciferase+allows+for+rapid+and+quantitative+testing+of+antivirals.">A novel Ebola virus expressing luciferase allows for rapid and quantitative testing of antivirals.</a> Antiviral Res. 99, 207-213 (2013).</li><li>Diallo, J. S., 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=A+high-throughput+pharmacoviral+approach+identifies+novel+oncolytic+virus+sensitizers.">A high-throughput pharmacoviral approach identifies novel oncolytic virus sensitizers.</a> Mol Ther. 18, 1123-1129 (2010).</li></ol>

The authors have nothing to disclose.

Burada açıklanan lusiferaz bazlı bir kolaylığı, hız, en az ekipman gerekir, ve nispeten düşük maliyetle de dahil olmak üzere, diğer mevcut yöntemlere göre bir çok avantaj sağlamaktadır. Bu kadar önemli bir katkı, bir seri seyreltme adım kaçınma olduğunu. Bununla birlikte bu protokolün seri seyreltme esaslı türevler, kesinlikle mümkün olan ve son zamanlarda, yüksek verimli bir anti-viral Ekran 11-lusiferaz sentezleyen Ebola titrelerini ölçmek için kullanıldı. Doğal olarak daha fazla zaman alıcı ve daha pahalı olsa da gerektiğinde, bu tür uyarlamalar viral ölçümü için daha fazla dinamik aralık sağlayabilir. , Yüksek verimli taramalara bağlamında, viral titreleri değerlendirmek için özellikle uygun olmasına ek olarak, bizim tek aşamalı bir lusiferaz dayanılarak viral ölçme yöntemi virüs çoğaltma durumunda, enfeksiyonlu viryon doğru tahminlerini oluşturur. Bundan başka, aynı deneyden sitotoksisite verileri ile birlikte bir parlaklık okumaları daha eldehedef hücreler üzerinde deney koşullarının etkisinin tam resmi, onkolitik virüs ve ilaç ekranlarının bağlamında özellikle kullanışlıdır. Burada gösterilen örnek kopyalayan Negatif tek iplikli RNA virüsü kullanır; Bununla birlikte, bu protokol birkaç küçük ayarlamalar ile protokol virüs çoğalmasını ve replike edici olmayan bir dizi şekilde ayarlanabilir. Bu, Vaccinia, HSV ve AAV (bakınız Şekil 5) gibi maddeler, DNA virüslerini içerir. Örneğin vaccinia virüsü gibi hücre içi virüs ile enfekte numuneler önce, miktar (örneğin, en az bir dondurma-çözme çevrimi) için bir virüs bırakma adımı gerektirir. Diğer virüsler için bu tekniği uygulandığında, lüminometre ile plakaların okuma viral süpernatan veya lizat transfer kuluçka süresinin optimize edilmesi gereklidir. Bu parametre izin veren hücre hattında virüsün replikasyon döngüsü ve p gücü esas bağlı olacaktırromoter lusiferaz ekspresyonunu tahrik. Bu en iyi duruma getirme aşamasında tam bir standart eğri kullanılarak yapılır. Bunu yapmak için, bir hazırlanan standart eğri çeşitli çoğaltır ile uygun müsamahakâr hücre hattı bulaştırmak ve post-devret işaret her bir farklı bir zamanda çoğaltmak okumak gerekir. İdeal olarak, bir kuluçka zaman noktası beklenen örnek titre aralığı kapsayan LOG (RLU), ve log (titre) arasında doğrusal bir ilişki yol açar seçilir. VSVΔ51-Fluc için, bu tipik olarak 4 ila 10 pfu / mL -10 7 pfu / ml, 5 saat inkübasyon süresi için. Düşük veya yüksek titreleri numunelerden beklenen varsa, biri sadece sırasıyla artırmak veya kuluçka süresini azaltabilir. Alternatif olarak, örnekler, ELISA için tipik olarak yapıldığı gibi daha aralığına düşecek şekilde seyreltilebilir. Yukarıda belirtildiği gibi, bu yöntem de en adımların otomatik edilebilir ilaç kütüphaneleri kullanılarak yüksek verimli ilaç ekranlar gerçekleştirmek için uygundur. Hücreler e kaplı olabilirfficiently otomatik bir mikro dağıtıcısı kullanılarak ilaç kütüphane 96 kanallı bir sıvı tutucu yolu ile eklenebilir, virüs bir mikro-dağıtıcı kullanılarak ilave edilebilir ve plakalar, bir otomatik luminometre kullanılarak okundu. Teorik olarak, bu da 384-kuyucuklu bir veya daha küçük formatlarda adapte edilebilir; Bununla birlikte, bu amaçla sınırlama kaplama olabilir hücre sayısı, verilen daha az hücre (titre) ilişki için kullanılacak günlük (RLU) arasında doğrusal olarak dar bir aralığı sebep olur. Son olarak, Resazurin ve diğer metabolik boyalar kullanılarak hücre canlılığı değerlendirmesi kolay virüs 12 ile kombinasyon halinde sinerjistik bir antiviral öldürülmesine yol ekranlar ya da bileşiklerin tanımlanması sitotoksik bileşiklerin ayrım sağlayan, iş akışı içinde dahil edilebilir. Bununla birlikte, bu yöntemin sınırlamaları her zaman mümkün değildir, bir lusiferaz transgen ifade virüs, gereksinimi ve yeterince izin veren hücre hattının durumunu içerir. Bununla birlikte, adapte etmek mümkündür muhtemeldirDiğer raportör gen ile birlikte kullanılmak için olan yöntem (örneğin GFP) raportör ölçme yöntemi gürültü oranı için uygun bir doğrusallık ve sinyal sahip olması koşuluyla. Genel olarak, anlatılan yöntem, yüksek verimli bir çok farklı virüs uyacak şekilde değiştirilebilir ve farklı uygulamalar için uygun olabilir.

Classical viral plaque assays continue to be a mainstay in virus research even though they can be notoriously time-consuming and constitute a significant bottleneck to obtaining results from experiments. More rapid indirect virus quantification methods have emerged including quantitative polymerase chain reaction (qPCR), ELISA, and flow cytometry1-4. Recent innovations such as the Virocyt virus counter can directly count viruses using advanced flow sorting technology and through a combination of protein and DNA/RNA dyes5. While all of these methods have undoubtedly quickened the pace of virus research, each method has its advantages and drawbacks. For example, qPCR can allow for quantification of specific viral genome sequence but cannot effectively discriminate infectious from defective virions6. ELISAs can be very specific however require a suitable antibody against the desired target viral protein and can be very expensive. While flow cytometry technology offers many advantages and has improved significantly, throughput and accessibility to highly specialized equipment nonetheless remains a hurdle. Importantly, all of these techniques are not ideally suited for high-throughput screening, for which the ease and time requirement of the virus quantification step is of critical importance.
Here we describe a high-throughput and easily automatable technique to titer viruses that express a Firefly luciferase (Fluc) transgene. This method generates approximate viral titers in a test sample based on luminescence signal reads through the parallel use of a standard curve of known amounts of virus. Samples containing unknown quantities of luciferase-expressing virus are transferred on to a permissive &#8220;plaquing&#8221; cell line in parallel with the standard virus dilution curve and virus-associated luminescence is read after a few hours incubation time. This allows for rapid, quantitative, often same-day generation of results, unlike classic plaque assay protocols which typically require several days of incubation in order to manually count visible plaques7-9. 
The protocol outlines the steps of our titration method using oncolytic Vesicular Stomatitis Virus encoding a Fluc transgene (VSV&#8710;51-Fluc) as an example and provides an overview of 1. Sample preparation 2. The plating of a permissive cell line for virus titration using an automated dispenser 3. The preparation of the viral standard curve 4. The transfer of the sample supernatants onto the permissive cell line using a 96-well manual pipettor 5. The assessment of sample cytotoxicity using a cell viability reagent 6. The preparation of the luciferin substrate 7. Reading of bioluminescence and 8. Data analysis.

<table border="0" cellpadding="0" cellspacing="0" width="808">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Name of Material/ Equipment</td>
			<td style="width:115px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td style="width:251px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbeccos&#39; Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Corning</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>NorthBio Inc.</td>
			<td>NBSF-701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Phosphate buffered saline</td>
			<td>Corning</td>
			<td style="width:136px;">21-040-CV</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">HEPES</td>
			<td style="width:115px;">Fisher Scientific</td>
			<td style="width:136px;">BP310-1</td>
			<td style="width:251px;">Prepare a&#160; 1mM solution, pH 7.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">alamarBlue&#160;</td>
			<td>AbD Serotec</td>
			<td>BUF012B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Luciferin potassium salt</td>
			<td>Biotium</td>
			<td>10101-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">96-well solid white flat bottom polystyrene TC-treated microplates</td>
			<td>Corning</td>
			<td align="right" style="width:136px;">3917</td>
			<td style="width:251px;">384-well plates can also be used for higher throughput</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synergy Mx</td>
			<td>BioTek</td>
			<td>SMTBL</td>
			<td>Monochromator microplate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liquidator96</td>
			<td>Mettler Toledo</td>
			<td>LIQ-96-200</td>
			<td>96 tip manual pipetting system</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Liquidator96 LTS Tips sterilized with filters</td>
			<td>Mettler Toledo</td>
			<td style="width:136px;">LQR-200F</td>
			<td style="width:251px;">Any sterile filtered tips compatible with pipettors of choice are appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microflo</td>
			<td>BioTek</td>
			<td>111-206-21</td>
			<td>Used to plate cells in a 96 well plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluoroskan Ascent FL</td>
			<td>Thermo Scientific</td>
			<td>5210450</td>
			<td>Microplate fluorometer</td>
		</tr>
	</tbody>
</table>

high-throughput titration of luciferase-expressing recombinant viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

Yüksek verimli bir metodu tarif eden akışı bir özeti Şekil 1 &#39;de gösterilmektedir. Şekil 2 tipik bir standart eğri ofVSVΔ51-Fluc sonuçlarını göstermektedir. Non-lineer regresyon analizi Dört-parametresi bilinmiyor giriş pfu (viral titreye tahmini) interpolasyon edilebileceği bir Tepesi arsa oluşturulur. Bu tahmini titerleri olarak adlandırılır viral sentezleme birimi (VEU). Şekil 3A ve Veus titreleri aynı örnekler 10, standart plak test sureti ile elde edilen göstermektedir. Numuneler çeşitli kimyasallar çoğaltma geliştirmek için kullanılabilir, 786-0 hücrelerinde VSVΔ51-Fluc arasında yayılmış olan bir deney kaynaklanmıştır. Standart eğri interpolasyon Veus örnek süpernatanların seyreltilmesine dayanan bir faktör ile çarpılmalıdır Vero hücreleri üzerine transfer edilmektedir (bu durumda, seyreltme faktörü 5&#39;tir). Titreleri ve VEU arasında doğrusal bir ilişki Şekil 3B&#39;de gösterilmiştir </strong&gt; Ar 0,8083 2 ve 0,899 (p &lt;0.0001) Pearson r skoru. Şekil 4&#39;te, bir metabolik tahlilinden elde edilen tipik bir hücre sitotoksisitesi veriler önceden VSVΔ51-Fluc enfeksiyonuna kimyasal ile muamele 786-0 hücreler için gösterilmiştir. Son olarak, Şekil 5, çeşitli virüs ile kullanılarak elde edilen standart bir eğri (herpes simpleks virüsü (HSV), vaccinia virüs ve adeno-ilişkili virüs (AAV), tüm ifade ateşböceği lüsiferaz) gösterir ve gerekirse, inkübasyon sürelerini ve donma-erime döngüleri tarif eder. <img alt="Şekil 1" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig1highres.jpg" width="500" /> VSV-Δ 51 Fluc yardımıyla yüksek verimli, virüs titre ve sitotoksisite tahlilinin Şekil 1. İş Akışı. 37 ° C&#39;de (A) Tohum oyuk (100 ul) başına 2.5 x 10 4 Vero hücreleri ve inkübe edilir. (B) 24saat sonra aktarım için Vero hücreleri üzerinde standart bir eğri 25 ul (plaka başına 2 sütun). (C) örneklerinin Aktarım 25 ul kalan Vero hücreleri üzerinde titre etmek için. Santrifüj plakaları ve de 37 ° C &#39;. (D) içindeki hacmin% 10&#39;una eşit olan bir miktar içinde en inkübe orijinal numune içeren plağa resazurin reaktifmi. , 5 saat sonra, (F). Sitotoksisite floresans okunur ve değerlendirilmesi, 3 saat sonra, 37 ° C&#39;de. (E) inkübe edin Vero hücrelerinin her oyuğuna luciferase 2 mg / ml çözeltisi 25 ul ekle. Luminescence okuyun ve Viral İfade Birimleri hesaplayın. <img alt="Şekil 2" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig2highres.jpg" width="500" /> Şekil 2. VSV Δ 51-Fluc standart eğri Beklenen. Lüsiferaz ifadesi bendimiyi bir luminometre kullanılarak ve bioluminesans ortalama bağıl ışık birimleri (RLU) olarak ifade edildi içinde beş farklı noktalarda 5 saat sonrası süpernatant transferi asured. RLU ortalama bilinen girişine karşı çizilmiştir pfu / ml non-lineer regresyon çözmek ve Hill-Denklemi üretir. Iki suret eğrileri ve standart hata çubukları ortalama (r = 0,9993 2) gösterilmiştir. <img alt="Şekil 3," fo:content-width="5in" src="/files/ftp_upload/51890/51890fig3highres.jpg" width="500" /> Şekil yüksek verimli bir yöntem ile elde edilen standart plak test titrelerinin 3. karşılaştırılması. (A), VEU / ml arasında pfu / mL yüksek verimli bir lusiferaz deneyi kullanılarak titre aynı örneklerden hesaplanan viral titreler (VEU / ml) ile karşılaştırıldı Vero hücreleri üzerinde standart plak deneyi ile elde edilmiştir. (B) lineer bir ilişki içinde viral titreleri ve titre standart p yoluyla elde edilenlaque deneyi. Doğrusal regresyon eğrisi ve kararlılıkla (R 2) katsayısı gösterilmiştir. <img alt="Şekil 4" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig4highres.jpg" width="500" /> Şekil 4. Örnek mevcudiyetini göstermektedir. Vero hücreleri üzerinde yüzer transferi, ticari olarak temin edilebilir çözelti kullanılarak Resazurin hücresel metabolik aktivitesinin değerlendirilmesi ile belirlenmiştir örnek canlılığı önce. Ham flüoresan değerleri, muamele edilmemiş, enfekte olmamış kuyusunun en az normalize edildi. <img alt="Şekil 5," fo:content-width="5in" src="/files/ftp_upload/51890/51890fig5highres.jpg" width="500" /> Şekil 5. HSV, vaksiniya, ve AAV virüs, standart eğriler beklenir. Lusiferaz tanımı, iyi bir luminometre kullanılarak bir mesafede olan beş farklı noktalarda ölçülmüştür ve biyoışıldama ortalama gerçek konusunda ifade edilditif ışık birimleri (RLU), (A). HSV standart bir eğri Vero hücrelerine ilave edilmiştir 17 saat sonrası Süpernatan transferi (R2 yapıldı oyuk (100 ul) ve lusiferaz ölçüm başına 2.5 x 10 4 hücre yoğunluğunda 24 saat önce kaplı = 0,9489, n = 1). , Hill denklemi bir lineer olmayan regresyon çözerek elde edilmiştir. (B), bir standart eğri, Vaccinia Vero hücrelerine ilave edilmiştir oyuk (100 ul) başına 2.5 x 10 4 hücrelerinin 24 saat önce bir yoğunluğa yerleştirildi ve 37 ° C&#39;de 2.5 saat boyunca kuluçkaya C, bundan sonra lusiferaz ölçümleri alınmıştır (R 2 = 0,9892). Lineer bir bağıntı lineer regresyon çözülmesi ile elde edilmiştir. (C), bir standart eğri, AAV insan akciğer kanser hücreleri (A549) üzerinde ilave edildi oyuk (100 ul) ve lusiferaz ölçüm başına 2.5 x 10 4 hücre yoğunluğunda 24 saat önce kaplı 24 saat sonrası enfeksiyon yapıldı. A Hill denklemi doğrusal olmayan regresyon çözme ile oluşturulmuştur. OrtalamaBeş kopya eğrileri ve standart hata sütunları gösterilmiştir (R 2 = 0,9926).

Watch this Scientific Journal Video about High-throughput Titration of Luciferase-expressing Recombinant Viruses at JoVE.com

High-throughput Titration of Luciferase-expressing Recombinant Viruses

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

Lusiferaz ifade eden rekombinant virüsler yüksek verimlilik Titrasyon

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

high-throughput-titration-of-luciferase-expressing-recombinant-viruses

Universidad San Sebastian

Research

JoVE Journal

Biology

12.8K Görüntüleme.  Ottawa Hospital Research Institute. This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

Video: High-throughput Titration of Luciferase-expressing Recombinant Viruses

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

Lignocellulosic Malzemeler için Yüksek verim Saccharification Testi

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

Biyoloji

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

Affinity etiketli Rekombinant Proteinler Yüksek throughput Arıtma

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

Yüksek verim SHAPE kullanarak RNA İkincil Yapısı Tahmin

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

Deniz Kestanesi Zigotlar Yüksek Randımanlı Microinjections

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

Üretilen Rekombinant Disülfid zengin Venom Proteinler Uygulanan Yüksek Randımanlı Sayısal Anlatım Tarama ve Arıtma<em&gt; E. coli</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Yüksek verim Uygulamaları Yetişkin Zebra Hearts Kurtarma

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Post-transkripsiyonel ayarlı genlerin kimyasal Modülatörler için yüksek verimli tarama

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

Yüksek Verimli<em&gt; Danio rerio</em&gt; Enerji Harcaması Deneyi

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.

In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections of mineralized tissues.

Yüksek Verim, Mineralize Dokuların Çoklu Resim Cryohistology

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious ...

Precise, High-throughput Analysis of Bacterial Growth

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

Quantitative evaluation of bacterial growth is essential to understanding microbial physiology as a systems-level phenomenon. A protocol for experimental manipulation and an analytical approach are introduced, allowing for precise, high-throughput analysis of bacterial growth, which is a key subject of interest in systems biology.

Bakteriyel büyüme kesin, yüksek üretilen iş analizi

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the ...