ABD Ulusal Bilim Vakfı Doğu Asya ve Pasifik Yaz Enstitüleri;; Amerika&#39;da Araştırma Bursu Programı Jeoloji Derneği&#39;nin ve Lisansüstü Araştırma ve Burs Mezunlar Hibeler Bu çalışma, ABD Ulusal Bilim Vakfı (EAR-0920299 ve EAR-0745808) hibe tarafından desteklenen Ohio Eyalet Üniversitesi. Biz onların anlayışlı yorum yapmak için editör ve iki anonim teşekkür etmek istiyorum.

1. MTB Koleksiyonu <ol><li> Magnetotactic bakteri toplamak için bir tatlı su sitede (MTB) karar verirken, genellikle yumuşak bir çamurlu tortu tabakası olan bir gölet veya yavaş hareket akışı ile başlamak en iyisidir. Bu gösteri biz Columbus Ohio State Üniversitesi (OSU), Ohio (ABD) kampüsünde Olentangy Nehri kenarında bir örnek toplanmıştır. Bu bizim gösteri için elverişli bir konumda iken, burada açıklanan protokol herhangi bir sucul yere uygulanabilir. Bu protokolde kullanılan malzemeler Tablo 1&#39;de bulunabilir. Su derinliği 10 ile 100 cm arasında bir konuma bulun. Böyle bir yerde, sen sediment açık, vida-top konteyner kullanarak üst-en tabaka toplamak gerekir. O bir buçuk katı kadar üçte biri ve tortu su ile geri kalan hacim ile doldurulana kadar kap içine tortu su ve kepçe. Su ile dolana kadar daldırılmış bir kapta muhafaza edin ve sonra sıkıca konteyner w kapITH onun vida-üst kapak. Bu tortu karıştırmak için gerekli değildir. Bir havlu ile kurulayın kabın dışını silin ve sonra laboratuvara numune alır. Bu sizin laboratuar geri örnek acele gerekmez. Biz laboratuvar onları geri getirmeden önce birkaç gün boyunca alanında plastik kaplarda MTB örnekleri bıraktım. Eğer alanında gölge ve serin bir yerde saklayın örnekleri olarak MTB sürece birkaç hafta ay için geçerli olmalı. </li><li> ÖRNEK laboratuarda sonra, kap gevşetmek ve buharlaşma miktarını azaltmak için konteyner kaplama bırakın. Karanlık bir odada, çekmece içinde oda sıcaklığında saklayın konteyner ya da tamamen alüminyum folyo ile kap kapağı. Tortu ve ince parçacıklar tamamen bir veya birkaç gün birkaç saat boyunca rahatsız edilmeden örnek bırakarak kabın dibine yerleşmek için izin verin. Bu tortu karıştırmak için gerekli değildir, MTB kesintisiz bir ortamı tercih ederler. Plastik kabın açık duvarlarEğer parçacıklar altına yerleşmiş olduğunu doğrulamanızı sağlar. Numunenin bağlı MTB aylarca örnek hayatta kalabilir. </li></ol> 2. MTB İzolasyon <ol><li> Eğer yaklaşık 1 cm sediment-su ara (Şekil 1A) Yukarıdaki plastik kabın yüzüne MTB, yer mıknatıslar yalıtmak için hazır olduğunuzda. Kabın dibinde tortu rahatsız etmemek için dikkatli olun. Karşı tarafında (Şekil 1A) üzerinde konteyner ve başka bir bar mıknatısın kuzey tarafında bir tarafında bir çubuk mıknatısın güney kutbu yerleştirin. Hemen hemen her mıknatıs bu tür bir manyetik karıştırma çubuğu ya da büyük soğutma mıknatıs olarak, kullanılabilir. Her şey tortu-su ara yukarıda doğru yükseklikte mıknatısları desteklemek için kullanılabilir. Bir karton ya da plastik kutu üst mıknatıs dayanma en iyi, ancak, aynı zamanda mıknatıs plastik kabın dış tarafına yapıştırılabilir olduğunu tespit ettik. Birkaç 30 dk bekleyinbakteriler için saat mıknatıs yüzmek. </li><li> Dikkatle güney kutbu çubuk mıknatıs (Kuzey yarımkürede toplanan numuneler için) konumunu yakın konteyner (Şekil 1A) içindeki suyu toplamak için steril bir pipet kullanın. Bu su güney kutup çubuk mıknatıs için çekici olmuştur MTB içermelidir. Daha sonra, bir kapiler yarış pisti daha fazla MTB zenginleştirmek için kullanılabilir. </li></ol> 3. MTB Racetrack <ol><li> Magnetotactic bakteriler ile örnek zenginleştirmek için, kılcal bir yarış pisti (Şekiller 1B ve 1C) gereklidir. Bu berrak plastik kap gelen hücreler izole önce yapılmış olması gerekir. </li><li> Bir yarış pisti yapmak için 5,75 inç (146 mm) cam Pasteur pipeti kullanın. Pipet üst kesmek için elmas kalem veya dosyayı kullanın, pipet uzunluğu önemli değildir, ancak bu suyun yaklaşık 1-2 ml içeren gerekir. Sonra, ucu eritmek için bir Bunsen beki kullanın, böylece(Şekil 1C) sızdırmaz olur. Elde edilen pipet bir açık uç ve bir kapalı ucu sahip olmalıdır. </li><li> Bu pistlerde birkaç sonra otoklav olun. Ayrıca, pamuk ve birkaç uzun metal iğnelerin otoklavlamayın gerekir. </li><li> Süzülmüş bir enjektöre bağlanmış uzun metal iğne kullanarak bir otoklavlanmış hipodroma Şekil 1A gösterilen sediment su arabiriminin yanında toplanan filtrelenmiş su numunesinin, ekleyin. Filtre gözenek boyutu su pisliği ve kirletici maddeleri ortadan kaldırmak için 0.22 mm olmalıdır. Bu kılcal cam içinde hiçbir hava kabarcığı olduğunu kesinlikle emin olmak için önemlidir. </li><li> Steril pamuk (Şekil 1B) ile pist alt takın. Uzaklıkta mühürlü ucu (Şekil 1C) 1 cm - 0.5 o yüzden yarış pisti mühürlü sonuna doğru pamuk itmek için metal iğne kullanın. </li><li> Steril bir pipet kullanarak, örnek MTB içeren su (bölüm 2.2) ekleyin rhazırlanmış bir MTB yarış pisti (Şekil 1B) eservoir (açık uçlu). </li></ol> 4. MTB Zenginleştirme <ol><li> Racetrack örnek sıvı ile doldurulduktan sonra, yatay bir yüzey üzerinde yan yatıyordu (örneğin, bir benchtop) ve yarış pisti kapalı ucu (Şekil 1B yanında (Kuzey yarımkürede) bir çubuk mıknatısın güney kutbu yerleştirin ve 1C). </li><li> Pamuk yoluyla göç MTB için 5 ila 30 dakika bekleyin. Sonra pistin ucuna yakın sıvı toplamak gerekir. Çok uzun bekliyorum kılcal ucu gibi diğer hareketli bakteri gibi kirleticileri, tanıtabilirsiniz. İsteğe bağlı olarak, pistin ucu görüntülemek ve MTB Racetrack&#39;in ucunda toplamak izlemek için ışık mikroskobu kullanabilirsiniz. Bu onu pamuk yumağı içinden geçirmek MTB ne kadar sürer belirlemek için izin verir. </li><li> Yavaşça pamuk tapa yakınındaki küçük bir çizik yapıp pistin sonuna koparmak için elmas bıçak kullanın. </li> &lt;li&gt; pistin ucundan sıvı kaldırmak için dar bir iğne (25 ya da 27 gauge) ile 1 ml şırınga kullanın. Bu, sıvı numune hemen zenginleştirilmiş MTB içermelidir. </li></ol> 5. Işık Mikroskobu ile MTB Gözlem <ol><li> Bir lamel üzerine zenginleştirilmiş MTB örnek bir damla (10-20 ul) yerleştirin. </li><li> Hızla yüzden bırak şimdi aşağı bakacak ve lamel asılı olduğu üzerine lamel çevirin. </li><li> Bir cam slayt üzerine oturan bir o-ring üzerine lamel yerleştirin. O-ring sonra biraz daha küçük bir çapı lamel (; Şekil 2, yaklaşık 1 cm) sahip olmalıdır. </li><li> Bir ışık mikroskobu sahneye bu asılı damla yerleştirin ve açılan bir kenarına odaklanmak. Ama asılı damla yöntemi (Şekil 2) için kullanmak zordur yağ, gerekmez; çoğu yüksek sayısal açıklık (örneğin, 0.93 NA) çünkü bir 60X kuru hedefi çok iyi çalışıyor. </li><li> Asılı d bir çubuk mıknatıs yakın güney ucunda yerleştirinrop ve MTB mıknatıs (Şekil 3) en yakın damla kenarına doğru göç etmeye başlayacaktır. Birkaç dakika içinde çok sayıda MTB damla kenar (Şekil 3) olmalıdır. Bakteri mıknatıs kutup geri tarafından manyetik olduğu kendine kanıtlamak ve daha sonra bakteri ters yönde yüzmek dikkate alınmalıdır. </li></ol> 6. Transmisyon Elektron Mikroskobu (TEM) tarafından MTB Gözlem <ol><li> Bakır ızgara üzerine zenginleştirilmiş MTB bir damla (~ 20 ul) yerleştirin ve bakteriler 10 dakika için yerleşmek için izin. </li><li> Temiz filtre kağıt parçası ile fazla su Wick. </li><li> İsteğe bağlı olarak, ızgara olumsuz% 2 uranil asetat,% 2 fosfotungstik asit, pH 7.2,% 2.5 ya da sodyum molibdat 7, 8, 9 ile boyanmış olabilir. Bu, hemen zenginleştirilmiş MTB ile ızgara inkübe sonra leke üzerine bir damla bakır ızgara yerleştirilmesiyle gerçekleştirilir. Negatif leke ile ızgara inkübe kez st bağlı olarak değişirain kullanılan ve sonra temiz filtre bir parça kağıt ile sıvı kapalı fitil. </li><li> Transmisyon elektron mikroskobu (TEM, Şekil 4) kullanılarak MTB gözlemleyin. Iş Burada anlatılan MTB Formvar stabilize ve karbon kaplı 200 mesh bakır ızgaralar (Ted Pella # 01800) adsorbe edilmiştir. Şebekelerini 10 dk için hücre süspansiyonu damla karbon tarafı aşağıya doğru yerleştirildi, ardından hemen 30 saniye süreyle bir su damlası üzerinde ızgara yerleştirilmesi ile bir kez yıkanmıştır. Boyanması için, ızgaralar saniye 5 dakika için 30 için% 2 uranil asetat (Ted Pella # 19481), bir damla üzerine yerleştirilir ve daha sonra tamamen filtre kağıdı parçası kullanarak kurutuldu. Izgaraları TEM 200KV yüksek açılı halkasal karanlık alan KÖK kullanarak 80kV veya FEI Tecnai F20 ya hiç bir FEI Tecnai Ruh kullanılarak analiz edildi. </li></ol>

<ol>
	<li>Blakemore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetotactic+bacteria.">Magnetotactic bacteria.</a> Science. 190, 377-379 (1975).</li><li>Blakemore, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetotactic+bacteria.">Magnetotactic bacteria.</a> Annual Reviews in Microbiology. 36, 217-238 (1982).</li><li>Bazylinski, D. A., Frankel, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Biomineralization+of+Magnetite+(Fe3O4)+and+Greigite+(Fe3S4)+in+a+Magnetotactic+Bacterium.">Controlled Biomineralization of Magnetite (Fe3O4) and Greigite (Fe3S4) in a Magnetotactic Bacterium.</a> Applied and Environmental Microbiology. 61, 3232-3239 (1995).</li><li>Lefevre, C. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=South-seeking+magnetotactic+bacteria+in+the+Northern+Hemisphere.">South-seeking magnetotactic bacteria in the Northern Hemisphere.</a> Science. 311, 371-374 (2006).</li><li>Wolfe, R., Thauer, R., 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+'capillary+racetrack'+method+for+isolation+of+magnetotactic+bacteria.">A 'capillary racetrack' method for isolation of magnetotactic bacteria.</a> FEMS Microbiology Letters. 45, 31-35 (1987).</li><li>Rodgers, F. G., Blakemore, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+structure+in+a+many-celled+magnetotactic+prokaryote.">Intercellular structure in a many-celled magnetotactic prokaryote.</a> Archives of Microbiology. 154, 18-22 (1990).</li><li>Moench, T. T., Konetzka, W., 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+novel+method+for+the+isolation+and+study+of+a+magnetotactic+bacterium.">A novel method for the isolation and study of a magnetotactic bacterium.</a> Archives of Microbiology. 119, 203-212 (1978).</li><li>Balkwill, D., Maratea, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+of+a+magnetotactic+spirillum.">Ultrastructure of a magnetotactic spirillum.</a> Journal of Bacteriology. 141, 1399-1408 (1980).</li><li>Lins, U., Freitas, F., 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=Simple+homemade+apparatus+for+harvesting+uncultured+magnetotactic+microorganisms.">Simple homemade apparatus for harvesting uncultured magnetotactic microorganisms.</a> Brazilian Journal of Microbiology. 34, 111-116 (2003).</li><li>Jogler, C., Lin, W., 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=Toward+Cloning+of+the+Magnetotactic+Metagenome:+Identification+of+Magnetosome+Island+Gene+Clusters+in+Uncultivated+Magnetotactic+Bacteria+from+Different+Aquatic+Sediments.">Toward Cloning of the Magnetotactic Metagenome: Identification of Magnetosome Island Gene Clusters in Uncultivated Magnetotactic Bacteria from Different Aquatic Sediments.</a> Applied and Environmental Microbiology. 75, 3972-3979 (2009).</li><li>Lin, W., Li, J., 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=Newly+Isolated+but+Uncultivated+Magnetotactic+Bacterium+of+the+Phylum+Nitrospirae+from+Beijing,+China.">Newly Isolated but Uncultivated Magnetotactic Bacterium of the Phylum Nitrospirae from Beijing, China.</a> Applied and Environmental Microbiology. 78, 668-675 (2012).</li><li>Li, J., Pan, Y., 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=Biomineralization,+crystallography+and+magnetic+properties+of+bullet-shaped+magnetite+magnetosomes+in+giant+rod+magnetotactic+bacteria.">Biomineralization, crystallography and magnetic properties of bullet-shaped magnetite magnetosomes in giant rod magnetotactic bacteria.</a> Earth and Planetary Science Letters. 293, 368-376 (2010).</li><li>Oestreicher, Z., Valerde-Tercedor, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetosomes+and+magnetite+crystals+produced+by+magnetotactic+bacteria+as+resolved+by+atomic+force+microscopy+and+transmission+electron+microscopy.">Magnetosomes and magnetite crystals produced by magnetotactic bacteria as resolved by atomic force microscopy and transmission electron microscopy.</a> Micron. 43, 1331-1335 (2012).</li></ol>

Çıkar çatışması ilan etti.

Magnetotactic bakteriler mutlaka her sucul ortamda 8 bulunmayan ancak ortaya çıktığında, onlar 100 sipariş üzerine bulunabilir - mililitrede 2 başına 1.000 hücreleri. Optik mikroskop kullanılarak MTB gözlemlemek için, yaklaşık 50 bakteri / numunenin 8 ml gerekir. Sizin örnek hiç veya az MTB varsa, o zaman ya da örnekleri toplamak veya sonraki bölümde ele tekniklerden birini veya birkaçını deneyin gerekir yeni bir çevresel sitesi seçmeniz gerekecektir. <p clas...

<table border="1"><tbody><tr><td> Ürün Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> Cam slayt </td><td> Fisher Scientific </td><td> S95933 </td><td></td></tr><tr><td> Cam Pasteur pipetler </td><td> Fisher Scientific </td><td> 13-678-6A </td><td></td></tr><tr><td> O-ring </td><td> Donanım mağaza </td><td></td><td></td></tr><tr><td> Kapak fişleri </td><td> Fisher Scientific </td><td> 12 542B </td><td></td></tr><tr><td> Çubuk mıknatıs </td><td> Fisher Scientific </td><td> S95957 </td><td></td></tr><tr><td> Konteyner </td><td> Herhangi </td><td></td><td> Herhangi plasten az 0,5 L tutabilir ve kapatılabilir ic veya cam kap </td></tr><tr><td> Pamuk </td><td> Herhangi </td><td></td><td></td></tr><tr><td> 60X kuru lens ile Mikroskobu </td><td> Zeiss </td><td></td><td> Bir 60X kuru lensi kesinlikle gerekli değildir, ama bu yağ kullanmadan yüksek NA verir </td></tr><tr><td> Elmas kalem </td><td> Fisher Scientific </td><td> 08-675 </td><td></td></tr><tr><td> 0.22 mm filtre </td><td> Fisher Scientific </td><td> 09-719C </td><td></td></tr><tr><td> 1 ml şırınga </td><td> Fisher Scientific </td><td> NC9788564 </td><td></td></tr><tr><td> Mikrosantrifüj tüpleri </td><td> Fisher Scientific </td><td> 02-681-320 </td><td></td></tr><tr><td> Formvar / Karbon200 örgü, bakır ızgaralar </td><td> Ted Pella Inc </td><td> 01800 </td><td></td></tr><tr><td> Uranil asetat </td><td> Ted Pella Inc </td><td> 19481 </td><td></td></tr><tr><td> Tecnai Ruh TEM </td><td> FEI </td><td></td><td></td></tr><tr><td> Tecnai F20 S / TEM </td><td> FEI </td><td></td><td></td></tr></tbody></table>

collection, isolation and enrichment of naturally occurring magnetotactic bacteria from the environment

Magnetotactic bakteriler (MTB) ilk başta Massachusetts (ABD) tuzlu bataklıklar toplanan sediment örnekleri 1975 1 tarif edilmiştir sucul mikroorganizmalardır. O zamandan beri MTB tüm dünyada 2 yerinden tabakalı su ve sediman-sütunlarında tespit edilmiştir. Her MTB ile ortak bir özelliği, hücre içi, zar bağlı manyetik magnetit nanokristaller (Fe 3 O 4) ve / veya greigite (Fe 3 S 4) ya da her ikisi de 3, 4, magnetosomes, yer almasıdır. Güney yarımkürede genellikle bir mıknatıs 3,5 kuzey ucunda çekiliyor ise Kuzey yarımkürede, MTB tipik bir bar mıknatısın güney ucuna çekilir. Çevresel numunelerden MTB izole etmek için çalışırken bu özellik kullanılabilir. MTB zenginleştirmek için en yaygın yollarından biri, bir doğal kaynak sediment ve su toplamak için net bir plastik kap kullanmaktırbir tatlı su birikintisi gibi. Kuzey yarımkürede, bar mıknatısın güney ucunda sadece sediman-su ara sediman Yukarıdaki kabın dışına karşı yerleştirilir. Bir süre sonra, bakteri bir pipet ile mıknatıs yakınında kabın içine çıkarılabilir ve daha sonra bir kılcal yarış pisti 6 ve bir mıknatıs kullanılarak daha da zenginleştirilmiş. Bir kez, zenginleştirilmiş bakteri asılı damla yöntemi kullanarak bir mikroskop lamı üzerine yerleştirilebilir ve bir ışık mikroskobu gözlenen veya bir bakır ızgara üzerine yatırılır ve transmisyon elektron mikroskobu (TEM) kullanılarak gözlenmiştir. Bu yöntemi kullanarak, izole MTB mikroskobik gibi yüzme gibi özelliklerini belirlemek için incelenebilir hücreleri, manyetik kristallerin şekli, magnetosomes sayısı, her hücrede magnetosome zincirlerinin sayısı, kompozisyonu flagella, hücre morfolojisi davranışları, tipi ve sayısı nanomineral kristaller ve intrasellüler vakuoller varlığı.

Bir mıknatıs çevresel örneklerde (Şekil 1A) bulunan magnetotactic bakteriler (MTB) izole etmek için kullanılan etkili bir araçtır. Bir kılcal racetrack (Şekil 1B) aynı zamanda çevresel numune içinde bulunan sigara magnetotactic mikroorganizmalar ayrılabilir bir pamuk yumağı içinden onları çekmek için MTB manyetik özelliklerini kullanır. <img alt="Şekil 1" src="/files/ftp_upload/50123/50123fig1.jpg"...

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Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Bu doğal sularda da uygulanabilir magnetotactic bakteriler (MTB) toplamak için bir yöntem göstermektedir. MTB izole ve bakteri doğal manyetizma yararlanır nispeten basit bir kurulumu kullanarak sediment örneklerinin zenginleştirilebilir. İzole MTB sonra ışık ve elektron mikroskobu kullanarak hem ayrıntılı olarak incelenebilir.

Doğal Çevre gelen Magnetotactic Bakteri meydana gelen toplanması, İzolasyon ve Zenginleştirme

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

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22.9K Görüntüleme.  The Ohio State University. Bu doğal sularda da uygulanabilir magnetotactic bakteriler (MTB) toplamak için bir yöntem göstermektedir. MTB izole ve bakteri doğal manyetizma yararlanır nispeten basit bir kurulumu kullanarak sediment örneklerinin zenginleştirilebilir. İzole MTB sonra ışık ve elektron mikroskobu kullanarak hem ayrıntılı olarak incelenebilir.

Video: Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (&gt;80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

İnsan Kanı StemCell Teknolojileri RosetteSep Kit ile gelen NK Hücreleri zenginleştirilmesi

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

Biyoloji

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

Amerikan Horseshoe Yengeç, Limulus Polyphemus&#39;un Kan Toplama

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

Insan serumundan küçük kodlayıcı olmayan RNA&#39;lar izolasyonu

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. Birds are widely used subjects in biological research and are ideal for studies involving repeated sperm samples. However, few resources are currently available for those wishing to learn how to collect and extract DNA from avian sperm. Here we describe cloacal massage, a gentle, non-invasive manual technique for collecting avian sperm. Although this technique is established in the literature, it can be difficult to learn from the available descriptions. We also provide information for extracting DNA from avian semen using a commercial extraction kit with modifications. Cloacal massage can be easily used on any small- to medium-sized male bird in reproductive condition. Following collection, the semen can be used immediately for motility assays, or frozen for DNA extraction following the protocol described herein. This extraction protocol was refined for avian sperm and has been successfully used on samples collected from several passerine species (Passer domesticus, Spizella passerina, Haemorhous mexicanus, and Turdus migratorius) and one columbid (Columba livia).

This protocol describes cloacal massage, a non-invasive manual technique for collecting semen from birds, and DNA extraction from avian sperm using a commercial extraction kit with modifications.

Kuş sperm toplama Cloacal masaj ve Sperm DNA izolasyonu

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. ...

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ability to biomineralize magnetosomes, which are magnetic nanometer-sized crystals of magnetite (Fe3O4) or greigite (Fe3S4) surrounded by a lipid bilayer membrane, within their cytoplasm. For most known magnetotactic bacteria, magnetosomes are assembled in chains inside the cytoplasm, thereby conferring a permanent magnetic dipole moment to the cells and causing them to align passively with external magnetic fields. Because of these specific features, magnetotactic bacteria have a great potential for commercial and medical applications. However, most species are microaerophilic and have specific O2 concentration requirements, making them more difficult to grow routinely than many other bacteria such as Escherichia coli. Here we present detailed protocols for growing three of the most widely studied strains of magnetotactic bacteria, all belonging to the genus Magnetospirillum. These methods allow for precise control of the O2 concentration made available to the bacteria, in order to ensure that they grow normally and synthesize magnetosomes. Growing magnetotactic bacteria for further studies using these procedures does not require the experimentalist to be an expert in microbiology. The general methods presented in this article may also be used to isolate and culture other magnetotactic bacteria, although it is likely that growth media chemical composition will need to be modified.

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

We present a procedure for growing several strains of Magnetospirillum in two different types of growth media. Magnetospirillum gryphiswaldense strain MSR-1 is grown in both liquid and O2 concentration gradient semi-solid media while M. magneticum strain AMB-1 and M. magnetotacticum strain MS-1 are grown in liquid medium.

Cins Magnetospirillummagnetotactic bakteri büyüyen: suşları MSR-1, AMB-1 ve MS-1

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

Yetişkin Murine lacrimal ve submandibular bezlerden Myoepitelyal hücrelerin izolasyonu

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Organoid Kültür için İnsan Akciğer Epitel Progenitör Hücrelerinin İzolasyonu ve Zenginleştirilmesi

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and&#160;seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for&#160;future pandemics.

A citizen science project was designed to recruit San Diego residents to collect environmental samples for SARS-CoV-2. A multilingual web-based platform was created for data submission using a user-friendly mobile device interface. A laboratory information management system facilitated the collection of thousands of geographically diverse samples with real-time outcome tracking.

Kentsel Çevrenin Taranması - Çevre Rezervuarlarından SARS-CoV-2'nin Örneklemesi ve Tespiti için Bir Boru Hattı

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Ökaryotik Hücrelerden Çeviri Komplekslerinin Hızlı In Vivo Fiksasyonu ve İzolasyonu

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

İnsan Osteoartrit Diz Ekleminden Doku Toplama ve RNA Ekstraksiyonu

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...