Bu eser desteklenen Fondo Nacional de Desarrollo Científico y Tecnológico (Fondecyt), Şili, (CW için) proje 11130263, proje CONICYT + NERC + Programa de tarafından Colaboración Internacional PCI-PII20150073 (için CW) ve U-inicia: Vicerrectoría de Investigación Universidad de Chile (CW için).
Tatiana Naranjo-Palma için Ciencias Químicas y Dr. Jorge Babul ve Diego Quiroga-Roger yararlı tartışma, el yazması ve Dirección de extensión de la Facultad de gözden geçirilmesi için Robert Lesch için bu projenin ilk aşaması yardım için teşekkür Universidad de Chile üzerinden Farmacéuticas. Marcelo video düzenleme için Pérez ve Neil Stevens ses sağlamak için metin ve Errol Watson düzeltmek için çok teşekkür ederiz.

1. hazırlanması ve jel çalıştıran
<ol><li>Sambrook ve Russell14 ' te bulundu ama su SDS yerine kullanarak Sodyum Lauryl Sülfat-Polyacrylamide Jel Elektroforez (SDS-sayfa) tarifi kullanarak sigara denaturing % 12 polyacrylamide jel jelleri hazırlayın.<ol>
<li>5 mL jel çözme hazırlamak için 1.7 mL distile su, 2 mL Akrilamid/BIS Akrilamid (%30/0,8 w/v), 1,5 M Tris pH 8.8 1.3 mL, 0.05 mL % 10 amonyum persülfat ve TEMED 0.002 mL kullanın.</li>
		<li>Yığın jel 2 mL hazırlamak için 1,5 mL su, Akrilamid/BIS Akrilamid (%30/0,8 w/v) 0.33 mL, 1 M Tris pH 6.8 0.25 mL, 0,02 mL % 10 amonyum persülfat ve TEMED 0.002 mL kullanın. 7,3 cm x 8,2 cm x 0.075 boyutlardır jel cm (l x g x d). Not: 15 iyi tarak 0,75 mm kalınlıkta en iyi duyarlılık için kullanın.</li>
	</ol>
</li>
	<li>Tampon ve DNA örneği 1.5 mL microcentrifuge Tube (örneğin 6 x yükleme arabelleği için DNA örneği 5 µL kullanım 1 µL) yükleme x 6 uygun miktarda ekleyebilirsiniz.</li>
	<li>Jel Dikey Elektroforez odasına koyun.<ol>
<li>Odası 2 jelleri çalıştırmak için 1 x TAE arabellek 750 mL ile doldurulması. 1 L 50 hazırlamak için x TAE, 442 g Tris tabanının, buzul asetik asitin 57.1 mL ve 100 mL 0.5 M EDTA pH 8 kullanın. 1 L. son bir hacim için distile su ilave</li>
		<li>Jel örneklerinde yükleyin. Jel 100 V 2 h için çalıştırın.</li>
	</ol>
</li>
	<li>Alternatif olarak, 1 x TAE özel jel Sambrook14' te bulunan tarifi kullanarak hazırlayın.<ol>
<li>1 300 mL ekleyin x TAE yatay Elektroforez odasına. 45 dk için jel 100 V çalıştırın.</li>
	</ol>
</li>
</ol>2. NBT-SG ben boyama (şekil 1).
<ol><li>
Akrilamid boyama jel.
	<ol>
<li>Çözümler hazırlamak: % 0,1 w/v NBT ve 1.2 x SG ben. Not: Etidyum bromür, GelRed ve SYBR altın SG yerine kullanmak mümkündür ama SG en iyi performansı vardı.</li>
		<li>1.2 16.75 mL dökün x SYBR çözüm uygun büyüklükte bir konteyner içine ben yeşil. Not: Kapsayıcı olarak bir pipet ucu kutusunun kapağı kullandık ve bu birimlerin bu kapsayıcı'ı kullanarak jel kapsayacak şekilde hesaplanır; onun boyutlardır 11,6 cm x 7,6 cm x 2.6 cm (l x g x d).</li>
		<li>Jel çözümde yerleştirin. 3.25 mL % 0,1 w/v NBT çözeltisi 0.2 mM NBT vermek ekleyin ve 1 x (2.0 µM için eşdeğer) SG ben son konsantrasyonları.</li>
		<li>Plastik film ile konteyner mühür ve tamamen kapsayıcı herhangi bir ortam ışığı engellemek için alüminyum folyo ile sarın. Not: Plastik film sıçramasına önlemek ve alüminyum folyo ile boyama çözüm reaksiyonu önlemek için kullanılır.</li>
		<li>Orbital Çalkalayıcı 60 RPM üzerinde 25 min için salla.</li>
		<li>Alüminyum ve plastik kapağı çıkarın. İçin 90 dk en fazla güneş ışığına maruz. Grubun ilgi görünene kadar her 5 dakikada kontrol edin.</li>
		<li>Mavi ışık kullanmak için bir protoboard 3 ışık - yayan diyot (LED) ile hazırlamak ve jel yüzey (sürekli olarak zaman boyama LED ışık kaynağı yoğunluğuna bağlıdır onay) 5 mm LED yerleştirin.</li>
	</ol>
</li>
	<li>
Özel boyama jel.
	<ol>
<li>Sonra hazırlama ve % 1'özel çalışan 1.4 bölümlerinde açıklandığı gibi jel (boyutlardır 1 cm x 6.2 cm x 7 cm; l x w x d), 2.1 Bu değişiklikleri ile olduğu gibi aynı adımları izleyin:</li>
		<li>1.2 41,9 mL dökün SG x ben çözüm içine kapağı bir pipet ucu kutusunun; onun boyutlardır 11,6 cm x 7,6 cm x 2.6 cm (l x g x d). 8.1 mL %0,1 NBT çözeltisi ekleyin.</li>
		<li>Plastik film ile konteyner mühür ve sonra tamamen kapsayıcı herhangi bir ortam ışığı engellemek için alüminyum folyo ile sarın.</li>
		<li>Orbital Çalkalayıcı 60 rpm'de 1 h için salla.</li>
	</ol>
</li>
</ol>3. veri analizi.
<ol><li>
NBT-SG ben kalibrasyon eğrileri.
	<ol>
<li>Jel görüntüleri 1200 dpi çözünürlükte tarama yoluyla elde edilir.</li>
		<li>Açık Yazılım görüntü analizi için. Bir resim editörü kullanarak görüntüleri düzleştirin. Bir görüntü Dansitometresi çalıştırın.</li>
		<li>Eğri altındaki alan hesaplamak. Bir grafik sinyal/sinyalMax DNA toplama karşı hazırlamak.</li>
	</ol>
</li>
</ol>4. DNA kurtarma.
<ol><li>1 ' % 1'özel jel hazırlamak tarifi kullanarak x TAE Sambrook14' te bulundu.</li>
	<li>1 300 mL ekleyin x TAE yatay Elektroforez odasına. 100 ng/µL pDJ10015 Plazmid ve Şekil 2' de verilen düzeni takip DNA merdiven 4 µL yükleyin. 1 h için jel 100 V çalıştırın.</li>
	<li>Mark ve iki 2 mL microcentrifuge tüp tartın.</li>
	<li>Jel iki özdeş yarısı için ortadan kesti. Her biri merdiven ve farklı boyama teknikleri karşılaştırmak için örnek plazmid bulunması gerekmektedir. Şekil 2' de şematik bir gösterimidir.</li>
	<li>Bir leke yarım NBT-SG ile ben olduğu gibi adım 2.<ol>
<li>Temiz bir neşter kullanarak plazmid bant kesme. Bir tüp jel parça kaydedin ve tartmak.</li>
	</ol>
</li>
	<li>Jel SG ile diğer parçası leke ben 1 50 mL ile SG x ben çözüm ve yavaş yavaş (60 rpm) 1 h için jel karanlıkta sallamak.<ol>
<li>Bir transilluminator jel yerleştirin ve temiz bir neşter kullanarak plazmid grup dışarı 2 dk. kesim için maruz.</li>
		<li>Diğer tüp jel parça kaydedin ve tartmak. Not: Protokol burada-20 ° C'de tüpler tutarken duraklatılmış</li>
	</ol>
</li>
	<li>Bir ticari jel ekstraksiyon kiti kullanarak plazmid ayıklama taşımak.</li>
</ol>5. plazmid normalleştirme.
<ol><li>1.5 mL microcentrifuge tüp ayıklanan plazmid 10 µL ve ben su türü 60 µL ekleyin. Mix ve transfer 60 µL kuvars küvet veya 2 µL doğrudan bir Nanodrop kullanın.</li>
	<li>Ölçmek soğurma spektrumları arasında 230-320 nm. Örnek'ın plazmid konsantrasyonu aşağıdaki formülü kullanarak hesaplar: <img alt="Equation 1" src="/files/ftp_upload/56528/56528eq1v3.jpg"> D uygulanan seyreltme faktörü nerede (Bu durumda 70/10 's), Ax x nm emme ve 50 µg/ml dsDNA için dönüştürme faktörü.</li>
	<li>9.1 ng/µL örnekleri sulandırmak.</li>
	<li>Escherichia coli (e.coli) yetkili hücreleri (örneğin XL10 altın) dönüştürmek 5 µL seyreltik örneği kullanarak Inoue dönüştürme yordamı16 takip.</li>
	<li>Koloniler Petri yemekler üzerinde saymak ve seyreltme unutmayın. Koloni oluşturan birimler (CFU) aşağıdaki formül17: ile hesapla <img alt="Equation 2" src="/files/ftp_upload/56528/56528eq2v2.jpg"> D ilk kültüründen uygulanan seyreltme faktörü nerede.</li>
	<li>Bir unpaired t-Testi gerçekleştirir.</li>
</ol>

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I., 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=Detection+of+DNA+using+a+visible+dye,+Nile+blue,+in+electrophoresed+gels.">Detection of DNA using a visible dye, Nile blue, in electrophoresed gels.</a> Anal. Biochem. 280 (2), 322-324 (2000).</li><li>Santill&#225;n-Torres, J. L. <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+stain+for+DNA+in+agarose+gels.">A novel stain for DNA in agarose gels.</a> Trends Genet. 9 (2), 40 (1993).</li><li>Adkins, S., Burmeister, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+DNA+in+agarose+gels+as+migrating+colored+bands:+applications+for+preparative+gels+and+educational+demonstrations.">Visualization of DNA in agarose gels as migrating colored bands: applications for preparative gels and educational demonstrations.</a> Anal. Biochem. 240 (1), 17-23 (1996).</li><li>Cong, W. 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R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: a Laboratory Manual. , (2012).</li><li>Hansen, W., Garcia, P. 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Yazarlar ifşa gerek yok.

Duyarlı ve roman yöntemi boyama DNA NBT azalma ve bu makalede sunulan SG kullanımı temel. Bu iletişim kuralı için kritik adım jel NBT-SG kuluçka zamanı ben çözüm ve NBT konsantrasyonu. Boyama için özel jelleri özel jelleri büyük kalınlığı nedeniyle Akrilamid tırnaklar için jeller daha uzun zamanı. NBT bu temas precipitates gibi bu yöntem de SDS huzurunda çalışmıyor. Jel mor bir arka plan alırsa, biz başka bir jel hazırlıklı olmak tavsiye ve ışık pozlama süresini azaltmak için. Eğe...

Biyokimya ve moleküler biyoloji geliştirmelerle DNA'yla çalışmaları daha iyi teknikleri DNA analiz etmek için gerekli. DNA boyama için belgili tanımlık ilk yöntem gümüş boyama, hangi çok hassas ama yoksun seçicilik ve örnek kurtarma izin vermez. Daha sonra floresan DNA boyama geliştirme örnek kurtarma imkanı ile DNA'ın miktar seçici izin. DNA miktar için kullanılan ilk floresan boyalar etidyum bromür1, mutajenik2olan biriydi. Ancak, şimdi orada daha güvenli ve daha hassas, GelRed ve SYBR yeşil gibi geliştirilmiş floresan boyalar ben (SG ben)3; Ama bütün bu floresan boyalar bir ultraviyole (UV) transilluminator veya fluorimeter kullanımı gerektirir.
DNA, metilen mavisi4 ve kristal violet5,6,7,8,9gibi gözle görülür boyama diğer tekniği vardır, ama tüm-in bunlar azaltılmış hassasiyeti muzdarip ve seçicilik. Tetrazolium tuzları organik bileşikler azaltılması için duyarlı ve bu durumda onlar çözünmez bir formu ve renkli formazan çökelti10,11. Son zamanlarda, bazı bivalent tetrazolium tuzları için DNA nedeniyle onların olumlu tetrazolium halkaları12bağlamak için gösterilmiştir.
Bir son yayın13' te, nitro mavi tetrazolium (NBT) ve floresan boyalar gibi etidyum bromür, GelRed denilen bir bivalent tetrazolium tuz azaltma kullanarak leke ve DNA polyacrylamide jeller ölçmek için yeni bir görünür teknik önerilmiş, SYBR yeşil ben ve SYBR altın. Mavi ışık veya güneş ışığı huzurunda çalıştı ve kalite ile örnek kurtarma izin bu reaksiyon ben ile UV transilluminator SG kullanımına kıyasla. Bu kağıt tetrazolium boyama tekniği detaylı bir protokol sağlar tuzlar hedefidir.

<table><tbody><tr><td>Nitro blue tetrazolium</td><td>Gold Biotechnology</td><td>298-83-9</td><td>reagent used in the staining</td></tr><tr><td>SYBR Green I 10000X</td><td>Thermo-Fisher</td><td>S7563</td><td>reagent used in the staining</td></tr><tr><td>Gel extraction kit</td><td>QIAGEN</td><td>28704</td><td>used to extract plasmid from the gel in integrity assay</td></tr><tr><td>DNA ladder</td><td>Thermo-Fisher</td><td>SM 1331</td><td>used to make calibration curves and as reference to cut band in integrity assay</td></tr><tr><td>Agar Agar</td><td>Merck</td><td></td><td>used to make LB-ampicillin culture plates&amp;nbsp;</td></tr><tr><td>sodium chloride</td><td>Merck</td><td>1064045000</td><td>used to make LB-ampicillin culture plates&amp;nbsp;</td></tr><tr><td>yeast extract</td><td>Becton, Dickinson and Company</td><td>212750</td><td>used to make LB-ampicillin culture plates&amp;nbsp;</td></tr><tr><td>peptone</td><td>Merck</td><td>72161000</td><td>used to make LB-ampicillin culture plates&amp;nbsp;</td></tr><tr><td>TEMED</td><td>AMRESCO</td><td>761</td><td>used to make polyacrylamide gels</td></tr><tr><td>ammonium persulfate</td><td>Calbiochem</td><td>2310</td><td>used to make polyacrylamide gels</td></tr><tr><td>acrylamide</td><td>invitrogen</td><td>15512-023</td><td>used to make polyacrylamide gels</td></tr><tr><td>bisacrylamide</td><td>SIGMA</td><td>146072</td><td>used to make polyacrylamide gels</td></tr><tr><td>Tris-base</td><td>Merck</td><td>1083821000</td><td>used to make TAE buffer and non-denaturing polyacrylamide gel tris-HCl buffer</td></tr><tr><td>EDTA</td><td>J.T. Baker</td><td>8993-01</td><td>used to make TAE buffer</td></tr><tr><td>Acetic acid</td><td>Merck</td><td>1,000,632,500</td><td>used to make TAE buffer</td></tr><tr><td>agarose</td><td>Merck</td><td>16802</td><td>used to make TAE buffer</td></tr><tr><td>spectrophotometer</td><td>Tecan</td><td>infinite 200 pro</td><td>used to quantify DNA plasmid</td></tr><tr><td>water purificatiom unit</td><td>Merck</td><td>Elix 100</td><td>use to make water type 1 (ASTM) used in spectrophotometry and DNA dilutions\</td></tr><tr><td>distilled water</td><td></td><td>-</td><td>used in gels, culture medium, stainings and general solutions</td></tr><tr><td>HCl</td><td>J.T. Baker</td><td>9535-03</td><td>used to make non-denaturing polyacrylamide gel tris-HCl buffer</td></tr><tr><td>6X DNA loading dye</td><td>Thermo-Fisher</td><td>R0610</td><td></td></tr><tr><td>5 mm Blue LED</td><td>Jinyuan electronics</td><td>SP-021</td><td>light source used in integrity assay</td></tr><tr><td>protoboard</td><td>KANDH</td><td>KH102</td><td>light source support and circuit&amp;nbsp;</td></tr><tr><td>9 V battery</td><td>Duracell</td><td>-</td><td>used to power the LED&#039;s</td></tr><tr><td>horizontal electrophoresis chamber</td><td>Biorad</td><td>1704486EDU</td><td>used to make and run agarose gel in integrity assay</td></tr><tr><td>vertical electrophoresis kit</td><td>Biorad</td><td>1658002EDU</td><td>used to run polyacrylamide gels in calibration curves</td></tr><tr><td>power supply</td><td>Biorad</td><td>1645050&amp;nbsp;</td><td>used to power the electrophoresis</td></tr></tbody></table>

dna staining method based on formazan precipitation induced by blue light exposure

DNA boyama yöntemleri Biyomedikal Araştırma için çok önemlidir. Biz DNA görselleştirme çıplak gözle renkli bir çökelti oluşumunu sağlayan basit bir yöntem tasarlanmıştır. Akrilamid ıslatarak çalışır veya özel DNA SYBR yeşil 1 x (2.0 µM için eşdeğer) bir çözümde jel ben (SG ben) ve bir mor üretir 0.20 mM nitro mavi tetrazolium güneş ışığına maruz formazan, çökelti veya özellikle mavi ışık. Ayrıca, DNA kurtarma testleri bizim yöntemi ile lekeli bir özel jel Ampisilin dayanıklı bir plazmid kullanılarak gerçekleştirilmiştir. Koloniler daha çok sayıda geleneksel ile bizim yöntemiyle elde edildi SG kullanarak boyama ben ultraviyole aydınlatmalı. Açıklanan yöntemi hızlı, belirli ve biomolecules bir transilluminator olmadan "çıplak gözle" görselleştirme izin DNA tespiti için toksik olmayan ve ucuz ve alan kullanımına uygun. Bu nedenlerden dolayı bizim yeni DNA'ları boyama yöntemi araştırma ve sanayi için potansiyel yararları vardır.

Mor formazan çökelti DNA olduğu (kütle spektrometresi13tarafından doğrulanmadı) görünür. Deneyde, bir DNA merdiven bir kalibrasyon eğrisi (şekil 3) yapmak için yüklendi. Protokol Akrilamid ve özel tırnaklar için jeller çalışır, ancak daha düşük bir şiddeti vardır ve özel daha uzun bir zaman alır. Ancak, piyasada bulunan bir seti kullanarak örnek kurtarma özel jelleri kullanılmasına ve ayıklama ile müda...

Watch this Scientific Journal Video about DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure at JoVE.com

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

Bu yöntem bir SYBR yeşil jel ıslatarak seçici boyama ve jelleri içinde DNA'ın miktar sağlar ben / Nitro mavi Tetrazolium çözüm ve sonra güneş ışığı ya da mavi bir ışık kaynağına maruz. Bu işlem görünür bir çökelti oluşturur ve alan kullanımı için ideal hale neredeyse hiçbir ekipman.

DNA Formazan yağış mavi ışığa maruz tarafından indüklenen esas alarak yöntemini boyama

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the ...

dna-staining-method-based-on-formazan-precipitation-induced-blue

Research

JoVE Journal

Biochemistry

9.4K Görüntüleme.  Universidad de Chile. Bu yöntem bir SYBR yeşil jel ıslatarak seçici boyama ve jelleri içinde DNA'ın miktar sağlar ben / Nitro mavi Tetrazolium çözüm ve sonra güneş ışığı ya da mavi bir ışık kaynağına maruz. Bu işlem görünür bir çökelti oluşturur ve alan kullanımı için ideal hale neredeyse hiçbir ekipman.

Video: DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, DNA is visualized by staining. This article demonstrates how to use thiazole orange to stain DNA. Thiazole orange compares favorably to common staining methods, in that it is sensitive, inexpensive, excitable with UV or blue light (to prevent sample damage), and safer than ethidium bromide. Labs already equipped to run DNA electrophoresis experiments using ethidium bromide can generally switch dyes with no additional changes to existing protocols, using UV light for detection. Blue-light detection to avoid sample damage can additionally be achieved with a blue-light source and emission filter. Labs already equipped for blue-light detection can simply switch dyes with no additional changes to existing protocols.

Here, we present a protocol to use thiazole orange for the detection of DNA in gel electrophoresis experiments. The use of thiazole orange allows elimination of ethidium bromide, and fluorescence detection can be achieved with either UV or blue light.

DNA Elektroforez tiyazol turuncu etidyum bromür veya alternatif boya yerine kullanma

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, ...

Biyokimya

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Memeli Hücreleri ile Derneği&#39;nin Bakterilerin Canlılık belirlenmesi için Floresan Mikroskopi Yöntemleri

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

İmmünoloji ve Enfeksiyon

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

Makrofaj Fagositoz ve Aktin Polimerizasyon Değerlendirme Yüksek Verimli Florimetrik Tekniği

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

Hücre içi sinyal iletimi Upconversion nanopartikül aracılı kinaz Photoactivation tarafından uzaktan tetiklemek için entegre bir sistem

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Biyomühendislik

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

Doğal Bileşik Kütüphaneleri Exploration uygundur Anti-biyofilm Tahliller Bir Platform

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Analysis of Lipid Droplet Content in Fission and Budding Yeasts using Automated Image Processing

Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are used as models in lipid metabolic research or for industrial lipid production. Lipid droplets are highly dynamic storage bodies and their cellular content represents a convenient readout of the lipid metabolic state. Fluorescence microscopy is a method of choice for quantitative analysis of cellular lipid droplets, as it relies on widely available equipment and allows analysis of individual lipid droplets. Furthermore, microscopic image analysis can be automated, greatly increasing overall analysis throughput. Here, we describe an experimental and analytical workflow for automated detection and quantitative description of individual lipid droplets in three different model yeast species: the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus, and the budding yeast Saccharomyces cerevisiae. Lipid droplets are visualized with BODIPY 493/503, and cell-impermeable fluorescent dextran is added to the culture media to help identify cell boundaries. Cells are subjected to 3D epifluorescence microscopy in green and blue channels and the resulting z-stack images are processed automatically by a MATLAB pipeline. The procedure outputs rich quantitative data on cellular lipid droplet content and individual lipid droplet characteristics in a tabular format suitable for downstream analyses in major spreadsheet or statistical packages. We provide example analyses of lipid droplet content under various conditions that affect cellular lipid metabolism.

Here, we present a MATLAB implementation of automated detection and quantitative description of lipid droplets in fluorescence microscopy images of fission and budding yeast cells.

Otomatik görüntü Işleme kullanarak Fission ve budding yeasts lipid damlacık Içeriğinin Analizi

Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are ...

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can be generated from FMN photolysis upon irradiation with visible light. The ROS generated from FMN photolysis are harmful to microorganisms, including pathogenic bacteria such as Staphylococcus aureus (S. aureus). This article presents a protocol for deactivating S. aureus, as an example, via photochemical reactions involving FMN under visible light irradiation. The superoxide radical anion (<img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" />) generated during the FMN photolysis is evaluated via nitro blue tetrazolium (NBT) reduction. The microbial viability of S. aureus that is attributed to reactive <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" /> species was used to determine the effectiveness of the process. The bacterial inactivation rate is proportional to FMN concentration. Violet light is more efficient in inactivating S. aureus than blue light irradiation, while the red or green light does not drive FMN photolysis. The present article demonstrates FMN photolysis as a simple and safe method for sanitary processes.

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Here, we present a protocol to inactivate pathogenic bacteria with reactive oxygen species produced during photolysis of flavin mononucleotide (FMN) under blue and violet light irradiation of low intensity. FMN photolysis is demonstrated to be a simple and safe method for sanitary processes.

Riboflavin-5′- Fosfatın Görünür Işık Fotolizi ile Patojenlerin İnaktivasyonu

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Floresan Antibiyotik Probları Kullanılarak Bakteriyel Direncin Görüntülenmesi

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

Temel işlemler sırasında mikrobiyal hücre büyümesini gözlemlemek için hücre Floresans mikroskobu canlı

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

Biyoloji

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid&#39;s position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

Hızlandırılmış Yapılandırılmış Aydınlatma Mikroskomu için Mitokondriyal Nükleoidlerin Özel Etiketlemesi

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...