Questo lavoro è stato sostenuto dalle concessioni dal US National Science Foundation (EAR-0920299 e EAR-0745808); US National Science Foundation dell&#39;Asia orientale e del Pacifico Estate Istituti, Geological Society of America del Programma di Ricerca Grant e le sovvenzioni per la ricerca Alumni Laurea e borse di studio da The Ohio State University. Vorremmo ringraziare l&#39;editore e due revisori anonimi per i loro commenti penetranti.

1. MTB Collezione <ol><li> Al momento di decidere su un sito per la raccolta d&#39;acqua dolce batteri magnetotactic (MTB), spesso è meglio cominciare con un laghetto o lento flusso che è uno strato morbido sedimento fangoso. In questa dimostrazione abbiamo raccolto un campione sul bordo del fiume Olentangy nel campus della Ohio State University (OSU) a Columbus, Ohio (USA). Mentre questo era una posizione comoda per la nostra dimostrazione, il protocollo qui descritto è applicabile a qualsiasi posizione acquatico. I materiali utilizzati in questo protocollo può essere trovata nella tabella 1. Trovare una posizione dove la profondità dell&#39;acqua è tra 10 e 100 cm. In tale posizione, si dovrebbe raccogliere più in alto strato di sedimento con una chiara, con tappo a vite contenitore. Raccogliere il sedimento e acqua nel contenitore fino a quando non è riempito con un terzo alla metà sedimenti e il restante volume con acqua. Mantenere il contenitore sommerso fino a riempirlo con acqua e poi ermeticamente tappare il contenitore with il suo tappo a vite del coperchio. Non è necessario mescolare il sedimento. Pulire l&#39;esterno del contenitore con un asciugamano e poi prendere il campione al laboratorio. Non è necessario correre il campione di nuovo al vostro laboratorio. Abbiamo lasciato i campioni MTB in contenitori di plastica nel campo per diversi giorni prima di portare di nuovo al nostro laboratorio. La MTB deve essere valida per diverse settimane o mesi, a condizione che si memorizzano i campioni in un luogo fresco e ombreggiato in campo. </li><li> Una volta che il campione è in laboratorio, allentare il tappo e lasciare che copre il contenitore per ridurre la quantità di evaporazione. Conservare il contenitore a temperatura ambiente in una stanza buia, cassetto o coprire completamente il contenitore con un foglio di alluminio. Permettono il sedimento e particelle fini di risolvere completamente il fondo del contenitore lasciando il campione indisturbato per diverse ore a diversi giorni. Non è necessario mescolare il sedimento, MTB preferiscono un ambiente indisturbato. Le pareti trasparenti del contenitore di plasticavi permetterà di confermare che le particelle hanno depositata sul fondo. A seconda del campione, MTB può rimanere in vita nel campione per molti mesi. </li></ol> 2. MTB isolamento <ol><li> Quando si è pronti per isolare i magneti, posto MTB sui lati del contenitore di plastica di circa 1 cm sopra il sedimento-acqua (Figura 1A). Attenzione a non disturbare il sedimento nel fondo del contenitore. Posizionare il polo sud di un magnete su un lato del contenitore e il lato nord di un altro magnete di barra sul lato opposto (Figura 1A). Quasi qualsiasi magnete può essere usato, ad esempio una barra di agitazione magnetica o magnete grande frigorifero. Tutto può essere usato per supportare i magneti alla giusta altezza sopra del sedimento-acqua. Abbiamo trovato che i magneti appoggiata sulla parte superiore di una scatola di cartone o plastica è meglio, tuttavia, i magneti possono anche essere incollata al di fuori del contenitore di plastica. Attendere 30 minuti a diverseore per i batteri a nuotare al magnete. </li><li> Utilizzare una pipetta sterile per raccogliere accuratamente l&#39;acqua all&#39;interno del contenitore (Figura 1A) vicino alla posizione del polo magnetico sud-bar (per campioni raccolti nell&#39;emisfero Nord). L&#39;acqua deve contenere la MTB che sono stati attratti dal polo sud-barra magnetica. Successivamente, una pista capillare deve essere utilizzato per arricchire ulteriormente la MTB. </li></ol> 3. MTB Racetrack <ol><li> Al fine di arricchire il vostro campione con batteri magnetotactic, una pista capillare è necessario (1B e 1C figure). Questi devono essere fatte prima di isolare le cellule dalla chiara del contenitore plastico. </li><li> Utilizzare un 5,75 pollici (146 mm) in vetro pipetta Pasteur per fare una pista. Utilizzare una penna diamante o file per tagliare la parte superiore della pipetta, la lunghezza della pipetta non è determinante, ma dovrebbe essere in grado di contenere circa 1-2 ml di acqua. Quindi, utilizzare un becco Bunsen per fondere la punta in modo chediventa sigillato (Figura 1C). La pipetta risultante dovrebbe avere una estremità aperta ed una estremità chiusa. </li><li> Fare alcuni di questi circuiti e poi autoclave. Inoltre, è necessario sterilizzare in autoclave cotone e molti aghi metallici lunghi. </li><li> Aggiungere acqua campione filtrato, raccolti dal vicino all&#39;interfaccia acqua sedimento mostrato in Figura 1A, ad una pista sterilizzato utilizzando un lungo ago di metallo attaccato ad una siringa filtrato. La dimensione dei pori del filtro dovrebbe essere 0,22 millimetri per eliminare i residui e contaminanti dall&#39;acqua. E &#39;importante essere assolutamente certi che non ci siano bolle d&#39;aria nel vetro capillare. </li><li> Inserire il fondo della pista con cotone sterile (Figura 1B). Utilizzare l&#39;ago metallico per spingere il cotone verso l&#39;estremità sigillata del circuito così è 0,5-1 cm dalla punta chiusa (Figura 1C). </li><li> Usando una pipetta sterile, aggiungere l&#39;acqua contenente MTB (dalla sezione 2.2) al campione reservoir (estremità aperta) di una pista preparata MTB (Figura 1B). </li></ol> 4. MTB Arricchimento <ol><li> Una volta che la pista è piena di fluido campione, si trovava su un lato su una superficie orizzontale (ad esempio, un banco) e posizionare il polo sud di un magnete (nell&#39;emisfero settentrionale) accanto alla punta sigillata della pista (Figure 1B e 1C). </li><li> Attendere 5 a 30 minuti per la MTB a migrare attraverso il cotone. Poi si dovrebbe raccogliere il fluido vicino alla punta della pista. Attendere troppo a lungo possibile introdurre contaminanti, come altri batteri motili, alla punta del capillare. Facoltativamente, è possibile utilizzare un microscopio ottico per vedere la punta del circuito e guardare la MTB raccogliere sulla punta del circuito di. Questo vi permetterà di determinare quanto tempo ci vuole la MTB a migrare attraverso il tappo di cotone. </li><li> Delicatamente usare il coltello di diamante per fare un piccolo graffio vicino tappo di cotone e far scattare la fine della pista. </li> &lt;li&gt; Utilizzare una siringa da 1 ml con ago sottile (25 o 27 gauge) per rimuovere il liquido dalla punta della pista. Questo campione di liquido dovrebbe ora contenere la MTB arricchito. </li></ol> 5. MTB osservazione al microscopio ottico <ol><li> Mettere una goccia (10-20 pl) del campione arricchito MTB su un vetrino coprioggetti. </li><li> Rapidamente capovolgere il coprioggetto sopra in modo che il calo è ora rivolta verso il basso e appeso alla coprioggetto. </li><li> Posizionare il vetrino su un o-ring che si riposa su un vetrino. L&#39;o-ring deve avere un diametro leggermente più piccolo allora il coprioggetto (circa 1 cm; Figura 2). </li><li> Posizionare questa goccia su un palco microscopio ottico e concentrarsi su un bordo della goccia. Un obiettivo 60X secco funziona molto bene perché hanno una più elevata apertura numerica (NA, ad esempio, 0,93) ma non richiedono l&#39;olio, che è difficile da usare per il metodo della goccia pendente (Figura 2). </li><li> Posizionare l&#39;estremità sud di un magnete vicino bar per l&#39;impiccagione dPOR e MTB inizierà a migrare verso il bordo della goccia più vicino al magnete (Figura 3). In pochi minuti MTB molti dovrebbe essere al bordo della goccia (Figura 3). Dimostra a te stesso che i batteri sono magnetici invertendo il polo del magnete e poi osservare i batteri nuotano nella direzione opposta. </li></ol> 6. Osservazione MTB da microscopia elettronica a trasmissione (TEM) <ol><li> Posizionare una goccia (~ 20 pl) della MTB arricchito su una griglia di rame e consentire ai batteri di depositarsi per 10 min. </li><li> Wick l&#39;acqua in eccesso con un foglio di carta filtro. </li><li> Facoltativamente, la griglia può essere negativamente colorate con acetato di uranile 2%, 2% pH acido fosfotungstico 7,2, o 2,5% di sodio molibdato 7, 8, 9. Questo viene fatto mettendo il reticolo di rame su una goccia di colorante immediatamente dopo incubazione della griglia con la MTB arricchito. Incubare la griglia con la macchia negativa, i tempi variano a seconda del stain utilizzato, e quindi favorire il liquido con un foglio di carta filtro. </li><li> Osservare la MTB con microscopia elettronica a trasmissione (TEM, Figura 4). Per il lavoro qui descritto MTB sono stati adsorbiti a formvar stabilizzato e di carbonio rivestiti 200 reti in rame maglia (Ted Pella # 01800). Le griglie sono stati collocati con il lato di carbonio su una goccia di sospensione cellulare per fino a 10 minuti, poi subito lavato una volta disponendo la griglia in una goccia di acqua per 30 sec. Per la colorazione, le griglie sono stati posti su una goccia di acetato di uranile 2% (Ted Pella # 19.481) per 30 sec per 5 minuti e poi asciugati completamente con un pezzo di carta da filtro. Le griglie sono state analizzate mediante TEM utilizzando uno Spirito FEI Tecnai a 80kV o FEI Tecnai F20 con alta STEM anulare angolo di campo scuro a 200kV. </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. T., Menguy, N., 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+Cultured+Greigite-Producing+Magnetotactic+Bacterium+in+a+Novel+Group+of+Sulfate-Reducing+Bacteria.">A Cultured Greigite-Producing Magnetotactic Bacterium in a Novel Group of Sulfate-Reducing Bacteria.</a> Science. 334, 1720-1723 (2011).</li><li>Simmons, S. L., Bazylinski, D. 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>

Nessun conflitto di interessi dichiarati.

Magnetotactic batteri non sono necessariamente presenti in ogni ambiente acquatico 8 ma quando si verificano, possono essere trovati dell&#39;ordine di 100 - 1000 cellule per millilitro 2. Al fine di osservare la MTB con microscopia ottica, avrete bisogno di circa 50 batteri / ml nel campione 8. Se ci sono pochi o nessun MTB nel campione, allora si avrà sia bisogno di selezionare un nuovo sito ambientale per raccogliere i vostri campioni o hai bisogno di provare una o più delle tecnich...

<table border="1"><tbody><tr><td> Articolo </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Diapositiva su vetro </td><td> Fisher Scientific </td><td> S95933 </td><td></td></tr><tr><td> Vetro Pasteur pipette </td><td> Fisher Scientific </td><td> 13-678-6A </td><td></td></tr><tr><td> O-ring </td><td> Ferramenta </td><td></td><td></td></tr><tr><td> Copertina scivola </td><td> Fisher Scientific </td><td> 12-542B </td><td></td></tr><tr><td> Bar magnete </td><td> Fisher Scientific </td><td> S95957 </td><td></td></tr><tr><td> Contenitore </td><td> Qualsiasi </td><td></td><td> Qualsiasi plastic o contenitore di vetro che può contenere almeno 0,5 L e possono essere sigillati </td></tr><tr><td> Cotone </td><td> Qualsiasi </td><td></td><td></td></tr><tr><td> Microscopio con obiettivo 60X a secco </td><td> Zeiss </td><td></td><td> Una lente 60X secco non è assolutamente necessario, ma questo dà un NA elevata senza utilizzare olio </td></tr><tr><td> Diamante penna </td><td> Fisher Scientific </td><td> 08-675 </td><td></td></tr><tr><td> 0,22 millimetri filtro </td><td> Fisher Scientific </td><td> 09-719C </td><td></td></tr><tr><td> 1 ml siringa </td><td> Fisher Scientific </td><td> NC9788564 </td><td></td></tr><tr><td> Provette da microcentrifuga </td><td> Fisher Scientific </td><td> 02-681-320 </td><td></td></tr><tr><td> Formvar / Carbon200 mesh, griglie di rame </td><td> Ted Pella Inc. </td><td> 01800 </td><td></td></tr><tr><td> Acetato di uranile </td><td> Ted Pella Inc. </td><td> 19481 </td><td></td></tr><tr><td> Tecnai Spirito 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

Batteri magnetotactic (MTB) sono microrganismi acquatici che sono stati in particolare di cui nel 1975 1 da campioni di sedimenti raccolti in saline di Massachusetts (USA). Da allora MTB sono stati scoperti in stratificato di acqua e sedimenti-colonne provenienti da tutto il mondo 2. Una caratteristica comune a tutti MTB è che contengono magnetosomes, che sono intracellulari, nanocristalli di membrana magnetici di magnetite (Fe 3 O 4) e / o greigite (Fe 3 S 4) o entrambi 3, 4. Nell&#39;emisfero settentrionale, MTB sono in genere attratti dal lato sud di un magnete, mentre nel Sud del mondo di solito sono attratti dal lato nord di un magnete da 3,5. Questa proprietà può essere sfruttata quando si cerca di isolare MTB da campioni ambientali. Uno dei modi più comuni per arricchire MTB è di usare un contenitore di plastica trasparente per raccogliere sedimenti e acqua da una fonte naturale,come uno stagno di acqua dolce. Nell&#39;emisfero settentrionale, all&#39;estremità sud di un magnete è posizionato contro la parte esterna del contenitore sopra il sedimento al sedimento-acqua. Dopo qualche tempo, i batteri possono essere rimossi dall&#39;interno del contenitore in prossimità del magnete con una pipetta e poi arricchito ulteriormente utilizzando un circuito capillare 6 ed un magnete. Una volta arricchito, i batteri possono essere posizionati su un vetrino da microscopio utilizzando un metodo goccia pendente e osservato in un microscopio ottico o depositato su una griglia di rame e osservato con microscopia elettronica a trasmissione (TEM). Usando questo metodo, isolato MTB possono essere studiate al microscopio per determinare caratteristiche quali nuoto comportamento, tipo e numero di flagelli, morfologia cellulare delle cellule, la forma dei cristalli magnetici, numero di magnetosomes, numero di catene magnetosome in ogni cella, la composizione di i cristalli nanomineral, e la presenza di vacuoli intracellulari.

Un magnete è uno strumento efficace che può essere utilizzata per isolare batteri magnetotactic (MTB) contenute in campioni ambientali (Figura 1A). Un circuito capillare (Figura 1B) utilizza le proprietà magnetiche di MTB attrarre attraverso un tappo di cotone dove possono essere separati dai non-magnetotactic microrganismi anche contenuti all&#39;interno del campione ambientale. <img alt="Figura 1" src="/files/ftp_upl...

Watch this Scientific Journal Video about Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment at JoVE.com

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

Dimostriamo un metodo per raccogliere batteri magnetotactic (MTB) che possono essere applicati alle acque naturali. MTB può essere isolato e arricchita da campioni di sedimento con una configurazione relativamente semplice che sfrutta il magnetismo naturale dei batteri. Isolato MTB può essere esaminata in dettaglio utilizzando sia la luce e la microscopia elettronica.

Raccolta, isolamento e di arricchimento di batteri naturalmente presenti magnetotactic dall&#39;ambiente

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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Biology

22.9K Views.  The Ohio State University. Dimostriamo un metodo per raccogliere batteri magnetotactic (MTB) che possono essere applicati alle acque naturali. MTB può essere isolato e arricchita da campioni di sedimento con una configurazione relativamente semplice che sfrutta il magnetismo naturale dei batteri. Isolato MTB può essere esaminata in dettaglio utilizzando sia la luce e la microscopia elettronica.

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.

Arricchimento di cellule NK dal sangue umano con il kit RosetteSep Technologies StemCell

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

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Biologia

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.

Raccolta di sangue dal Granchio Ferro di Cavallo Americano, Limulus Polifemo

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.

Isolamento dei Piccoli RNA non codificanti dal siero umano

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.

Raccolta dello sperma aviaria Cloacal massaggi e isolamento di DNA da sperma

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.

Crescita di batteri magnetotattici del genere Magnetospirillum: ceppi MSR-1, AMB-1 e 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.

Isolamento delle cellule Miepiteliali da adulti murina lacrimale e ghiandole sottomandibolari

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.

Isolamento e arricchimento di cellule progenitrici epiteliali polmonari umane per la coltura di organoidi

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.

Tamponare l'ambiente urbano - Una pipeline per il campionamento e il rilevamento della SARS-CoV-2 dai bacini ambientali

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.

Rapida fissazione in vivo e isolamento dei complessi traslazionali dalle cellule eucariotiche

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.

Raccolta dei tessuti ed estrazione dell'RNA dall'articolazione del ginocchio osteoartritico umano

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