Questo studio è stato sostenuto dal United States Department of Agriculture, Agricultural Research Service. I nomi commerciali o prodotti commerciali in questa pubblicazione sono citati unicamente allo scopo di fornire informazioni specifiche e non implica raccomandazione o approvazione da parte del Dipartimento dell&#39;Agricoltura degli Stati Uniti. USDA è un fornitore di pari opportunità e datore di lavoro.

1. La periferica Fringe di batterica Colony <ol><li> Grow X. fastidiosa (Xf) Temecula selvaggio di tipo 22, Pell delezione mutante Xf ΔpliG (utilizzando descritto in precedenza strategia di eliminazione 23), e il suo complementare XfΔpliG -C (utilizzando descritto in precedenza strategia di complementazione genetica cromosomica basata su 24) su PD2 piastre di agar 25 a 28 ° C per 5-7 giorni. </li><li> Cellophane Autoclave (1 x 1 cm 2) in acqua a 121 ° C (249 ° F) per 15 min. Pick up un pezzo di cellophane, scaricare l&#39;acqua toccando un angolo di cellophane su un piatto vuoto Petri, adagiare il cellophane oltre il 15% della superficie agar e asciugare all&#39;aria. </li><li> Pick up individuale X. colonie fastidiosa con sterile stuzzicadenti arrotondati e le cellule del punto in modo asettico su un foglio di cellophane sterilizzato sovrapposto sul 15% della superficie agar in piastre di agar. Incubare la piastras a 28 ° C per 2-3 giorni. </li><li> Esaminate la morfologia bordo delle colonie utilizzando un microscopio da dissezione con una lente obiettivo 2X e una lente oculare 10X. Fotografare la frangia periferico intorno colonie. </li></ol> 2. camere di flusso e microscopia microfluidici <ol><li> Fabbricare dispositivi microfluidici che utilizzano procedure di foto-litografico simili a quelli precedentemente descritti 5,18. Design a quattro canali paralleli con il software di progettazione assistita da computer su wafer di silicio master con i metodi litografici standard di 26. </li><li> Creare le camere di microfluidica dal maestro wafer di silicio con polidimetilsilossano (PDMS). Versare PDMS polimerizzate sopra master wafer di silicio e curare a 60 ° C per 1 ora. Staccare la replica PDMS dal master di wafer e tagliare la replica PDMS con una lama in un 22 mm x 40 mm come la stessa dimensione di un vetrino di vetro. </li><li> Esporre la replica PDMS, un vetrino di vetro (22 x 40 mm 2), ed un microscscivolo OPE (51 x 76 mm 2) per il plasma aria a 30 W per 2 min 27. Sandwich il corpo PDMS tra il vetrino di vetro e il microscopio di vetro per costruire la camera di uidic fl micro. </li><li> Praticare un foro (5,5 mm di diametro) attraverso le PDMS a ciascuna estremità del canale fantasia. Tagliare il tubo di gomma di silicone in lungo 12-20 cm. Inserire un&#39;estremità del tubo di gomma di silicone (5,1 mm di diametro esterno 2,1 mm diametro interno 0,8 mm parete) in ciascuna estremità dei canali della replica PDMS apertura e sigillare con PDMS polimerizzate a 60 ° C per 1 ora. </li><li> Collegare un&#39;altra estremità del tubo al fine spinato di connettori luer plastica. Avvolgere le camere microfluidica assemblati con il foglio di alluminio e autoclave per 20 min. </li><li> Raccogliere batteri cellule di X. fastidiosa wild type, mutante Xf ΔpliG, e complementare XfΔpliG -C tramite raschiatura, utilizzando inoculo monouso loop da PD2 piatti di media. Regolare la densità cellularead una densità ottica di 0,05 a 600 nm in PD2 brodo come descritto in precedenza 23. Raccogliere la soluzione di cellule batteriche in una tenuta di gas siringa da 1 ml. </li><li> Montare il dispositivo a microfluidi su un palco microscopio invertito. Collegare un tubo di ingresso alla siringa a tenuta di gas da 5 ml contenente PD2 brodo. Montare il 5 ml a tenuta di gas siringa con le pompe a siringa. </li><li> Collegare il tubo di scarico ad un serbatoio dei rifiuti. Mantenere una velocità media di flusso di 0,2 ml min -1 per 30 minuti per stabilizzare il sistema. </li><li> Collegare i tubi laterali di ingresso ad un 1 ml a tenuta di gas siringa contenente la soluzione cellula batterica. Lavare la soluzione cellula batterica attraverso il tubo di gomma fino a raggiungere il canale. Mantenere una portata media di 0,2 ml min -1 per altri 30 minuti, al fine di svuotare le cellule non legato dalla camera prima di cattura delle immagini. </li><li> Montare l&#39;otturatore microscopio sotto la parte di campo-adjusted del microscopio per controllare la luce. Collegare l&#39;otturatore per l&#39;otturatore coSistema ntrol e collegare il sistema di controllo dell&#39;otturatore al computer. </li><li> Montare una fotocamera digitale alla porta video del microscopio e collegarlo al computer. Eseguire il software di registrazione time-lapse, selezionare la funzione &quot;scatto&quot; dal menu, e riconoscere automaticamente l&#39;otturatore installato di default nel software per stabilire connessioni a l&#39;otturatore con il software. </li><li> Selezionare la funzione &quot;fotocamera digitale&quot; dal menu del software di registrazione time-lapse di riconoscere automaticamente la fotocamera digitale come dispositivo di acquisizione di default nel software e stabilire la comunicazione con la fotocamera digitale con il software. </li><li> Individuare le cellule batteriche in uno dei canali che utilizzano 20X ottica a contrasto di fase, quindi passare alla lente obiettivo 40X prima di cattura delle immagini. </li><li> Eseguire il software di registrazione time-lapse, selezionare la funzione &quot;acquisizione delle immagini&quot; utilizzando parametri di default dal menu per acquisire le immagini del microscopio.Quindi, aprire la funzione &quot;Acquisire time-lapse&quot; e impostare l&#39;intervallo di tempo di 30 sec 5,18,28 per la durata di 6-24 ore a seconda esperimento necessario per osservare spasmi motilità di X. fastidiosa 5,18,28. Fai clic su &quot;OK&quot; per avviare la registrazione time-lapse. Fai clic su &quot;Funzione Stack&quot; dal menù, selezionare &quot;Salva con nome&quot; per impilare le immagini nella cartella di destinazione sul computer dopo aver terminato la registrazione. </li><li> Per più canali, catturare immagini time-lapse dal primo canale ogni 30 sec per 6 ore. Spostare la lente dell&#39;obiettivo del microscopio al canale successivo per individuare le cellule bersaglio. Ripetere la funzione di time-lapse, come descritto sopra per catturare immagini in ciascuno dei quattro canali in sequenza se esperimento è configurato per utilizzare quattro canali. Continuare l&#39;acquisizione di immagini time-lapse per fino a tre giorni consecutivi. Tutti gli esperimenti sono stati condotti a temperatura ambiente (23 ± 2 ° C). </li><li> Compilare le immagini time-lapse in unfile video utilizzando il software di imaging di registrazione time-lapse. Eseguire il software di registrazione time-lapse, fai clic su &quot;funzione di Stack&quot; dal menu, e selezionare &quot;la funzione stack open&quot; per aprire i file impilati dal computer. </li><li> Avviare la funzione &quot;Crea filmato&quot; dal modulo pila, selezionando le immagini e scegliere il formato di output &quot;AVI&quot;. Fare clic su &quot;salva con nome&quot; per salvare il file video nella cartella di destinazione sul computer. </li><li> Selezionare i filmati raccolti dalla cartella di destinazione sul computer e riprodurli. Poi, osservare la motilità delle singole celle attraverso la visualizzazione risultante dell&#39;attività spasmi motilità delle cellule batteriche nei file video generati. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari concorrenti.

In questo studio, abbiamo caratterizzato il comportamento movimento di X. fastidiosa Pell mutante Xf ΔpilG ei suoi complementari ceppi XfΔpilG- C a nuova concezione multipla parallelo-nano-canale micro camere di fl uidic. Le camere uidic di nuova progettazione micro fl possono avere fino a quattro camere parallele con 100 micron nano-canale in larghezza rispetto a modelli precedenti con un solo 50 micron di larghezza di canale 18. Il miglioramento più ampio nano...

Xylella fastidiosa è un batterio Gram-negativo non flagellated, patogeno che provoca una serie di malattie economicamente importanti colture, compreso il morbo di Pierce a vite (Vitis vinifera L.) 1,2, 3. Questo batterio è limitata alla xilema conducibilità dell&#39;acqua vasi. L&#39;infezione di vite provoca il blocco dei vasi xilematici e si traduce in stress idrico e carenze nutrizionali 3. Colonizzazione successo dipende dalla capacità del batterio di muoversi dal sito iniziale di infezione per il resto dell&#39;impianto 3. Contrazione motilità è un mezzo di movimento batterica flagellare-indipendente, tramite l&#39;estensione, l&#39;attaccamento, e la scomparsa della tipo polare IV pili 4 che è stato caratterizzato X. fastidiosa 5,6,7. La motilità contrazione è stato osservato da pinzette laser e microscopia a forza atomica (AFM) 8,9,10. Utilizzando queste tecniche, tmotilità delle streghe generati dal tipo IV pilus di N. gonorrhoeae e P. aeruginosa sono stati caratterizzati da fl pili etichettatura uorescently e catturare i loro movimenti al microscopio. Sebbene entrambi i metodi hanno dettagliato la forza adesiva dei singoli batteri, le procedure sono complicato e lungo 9,10. I micro camere uidic fl sono stati usati per osservare la migrazione a lunga distanza delle singole cellule, così come piccoli aggregati di cellule batteriche 5,6. Queste camere sono state progettate come un microfabbricazione-nano-canale in un piatto integrato con un sistema di registrazione di immagini time-lapse 11,12,13,14. Micro fl dispositivi da camera uidic offrono diversi vantaggi per lo studio delle interazioni comportamentali movimento e cellula-cellula di batteri: (i) fornisce una piattaforma integrata con molteplici funzionalità del canale; (Ii) le spetta esaminare i movimenti e le aggregazioni di singole cellule nelle caratteristiche nano-scala di batteri; (Iii) permette di m direttaicroscopic registrazione delle immagini delle cellule batteriche e analisi time-lapse, (iv) fornisce a lungo termine osservazioni spaziali e temporali di singoli e / o popolazioni di batteri in un micro-ambiente; (V) la portata del mezzo di coltura in un canale può essere controllata con precisione è richiesta (vi) solo un piccolo volume (1 ml) di mezzo di coltura per ciascun esperimento. Di recente, il micro fl uidic flusso sistema è stato impiegato per studiare i comportamenti di cellule batteriche in diversi microambienti 14,15,16. L&#39;adesività e l&#39;attaccamento superficie E. coli 15, X. fastidiosa 16, e Acidovorax citrulli 14 alle superfici di vetro sono stati valutati utilizzando micro fl camere uidic. La formazione di aggregazione e bio fi lm mediato dal tipo IV pili di Acidovorax citrulli sono stati analizzati 14. Inoltre, il movimento di A. citrulli osservato sotto flusso cONDIZIONI hanno dimostrato che il pili di tipo IV può svolgere un ruolo importante nella colonizzazione e la diffusione di A. citrulli in vasi xilematici sotto alburno flusso condizioni. I motilità contrazione del Pseudomonas aeruginosa e X. cellule fastidiosa sono state osservate con successo contro una corrente di fluido in una camera di flusso microfabbricazione 5,6,17. Tipo IV pilus carente pilB e pilQ mutanti di X. fastidiosa sono stati trovati di alterare profondamente la velocità di spasmi motilità nelle condizioni di fl usso in Micro Devices uidic fl 5,6,18. Gli studi condotti su adesione batterica e la motilità in micro dispositivi uidic fl indicato che i micro fl camere uidic sono particolarmente adatti per l&#39;analisi della motilità contrazioni e la migrazione dei batteri pili mediata in vitro. Questi risultati spiegano il meccanismo di migrazione spasmi-mediata che facilita l&#39;attaccamento cellula-cellula, l&#39;aggregazione e la colonizzazione all&#39;internoil padrone di casa, alla fine portano a infezioni sistemiche. Pil-Chp operone di X. fastidiosa contiene Pell, Pili, pilJ, pillola, chpB e CHPC quale segnale di trasduzione codifica Percorsi di 20. I chemocettori transmembrana legano stimoli chimici nel dominio periplasmic e attivare una cascata di segnali nella loro porzione citoplasmatica di controllare in ultima analisi batterica motilità spasmi. Nel operone Pil-Chp di X. fastidiosa, una proteina Pell fosfo-navetta è un omologo di CheY. In E. coli e P. aeruginosa, CheY è il regolatore di risposta in sistemi chemiotassi che interagiscono con le proteine ​​motrici flagelli 19, 21. Anche se i contributi di dell&#39;operone Pil-Chp verso virulenza in X. fastidiosa stati esaminati recentemente 20, il ruolo di Pell in chemiotassi operone in risposta ai segnali ambientali e regolata / motore tipo IV pili di X. fastidiosa è uncLear. Per chiarire l&#39;intuizione di regolatore di chemiotassi Pell nell&#39;attività di spasmi motilità di X. fastidiosa, una camera uidic fl micro è utilizzato per valutare la motilità contrazioni di X. fastidiosa. La Pell di X. fastidiosa è caratterizzata confrontando i fenotipi di una delezione mutante Xf ΔpliG, ceppo complementare XfΔpliG -C e la sua wild type in vitro. I risultati evidenziano il ruolo di Pell nella motilità contrazioni di X. fastidiosa.

<table>
	<tbody>
		<tr>
			<td>Biology materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X. fastidiosa (Xf) Temecula wild type</td>
			<td></td>
			<td></td>
			<td>Costa, H. S., et al., 2004 22</td>
		</tr>
		<tr>
			<td>pilG deletion mutant Xf&#916;pliG</td>
			<td></td>
			<td></td>
			<td>Shi, X. Y., et al., 2007 26</td>
		</tr>
		<tr>
			<td>pilG complementary strain Xf&#916;pliG-C&#160;</td>
			<td></td>
			<td></td>
			<td>Davis, M. J., wt al. 1998 23</td>
		</tr>
		<tr>
			<td>Physical materials and equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable inoculating loops</td>
			<td>VWR international, Radnor, PA</td>
			<td>#22-363-607</td>
			<td>quantitative procedures such as bacterial collection</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning Corporation</td>
			<td>#0002709226</td>
			<td>Sylgard 184 silicone Elastomeric Kits</td>
		</tr>
		<tr>
			<td>AmScope MD2000 digital camera</td>
			<td>AmScope, Irvine, CA</td>
			<td>SE305R-AZ-E</td>
			<td>Image, video recording and measurement&#160;</td>
		</tr>
		<tr>
			<td>Tubes line</td>
			<td>Edgewood, NY</td>
			<td>#T4300</td>
			<td>Connected to the syringe and microfluidic chamber</td>
		</tr>
		<tr>
			<td>Plastic luer connectors</td>
			<td>Edgewood, NY</td>
			<td></td>
			<td>Connected to the syringe and microfluidic chamber</td>
		</tr>
		<tr>
			<td>Syringe pumps</td>
			<td>Pico Plus, Harvard Apparatus, MA</td>
			<td>#702209</td>
			<td>The flow rate can be adjusted while the pump is running.</td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>Gastight, Hemilton Company, Reno, NV</td>
			<td>#1005</td>
			<td>Provide the flowing broth</td>
		</tr>
		<tr>
			<td>Inverted Olympus IMT-2 microscope</td>
			<td>Olympus</td>
			<td>IMT-2 FLuoro PHase</td>
			<td>Image observation and recording</td>
		</tr>
		<tr>
			<td>SPOT-RT digital camera</td>
			<td>Diagnostic Instruments, Inc., MI</td>
			<td>RT230</td>
			<td>Image, video recording and measurement</td>
		</tr>
		<tr>
			<td>Microscope Shutter</td>
			<td>The UNIBLITZ, US</td>
			<td>#LS2T2</td>
			<td>Control camera&#8217;s exposure time</td>
		</tr>
		<tr>
			<td>Microscope Shutter Control system</td>
			<td>The UNIBLITZ, US</td>
			<td>VCM-D1</td>
			<td>VCM-D1 Single Channel CE/UL/CSA Approved Shutter Driver</td>
		</tr>
		<tr>
			<td>MetaMorph Image software</td>
			<td>Universal Imaging Corp., PA</td>
			<td></td>
			<td>Real-time super-resolution image processing&#160;</td>
		</tr>
	</tbody>
</table>

visualization of twitching motility and characterization of the role of the pilg in xylella fastidiosa

Xylella fastidiosa è un batterio non flagellated Gram-negativi che provoca una serie di malattie economicamente importanti delle piante. La motilità spasmi fornisce X. fastidiosa un mezzo per il movimento a lunga distanza intra-impianto e la colonizzazione, contribuendo verso patogenicità in X. fastidiosa. La motilità contrazioni di X. fastidiosa è gestito da pili di tipo IV. Tipo IV pili di Xylella fastidiosa sono regolati da Pell, un regolatore di chemiotassi in Pil-CHP proteine ​​di codifica operone che sono coinvolti con le vie di trasduzione del segnale. Per chiarire i ruoli di Pell nella motilità contrazioni di X. fastidiosa, un Pell -carente mutante Xf ΔpilG e il suo sforzo complementare XfΔpilG- C contenente nativo Pell sono stati sviluppati. A camere di microfluidica integrati con un sistema di registrazione di immagini time-lapse è stato utilizzato per osservare spasmi motilità in XfΔpIlg, XfΔpilG- C e il suo ceppo wild-type. Usando questo sistema di registrazione, permette a lungo termine osservazioni spaziali e temporali di aggregazione, la migrazione delle singole cellule e delle popolazioni di batteri via spasmi motilità. X. fastidiosa wild type e complementare ceppo XfΔpilG- C hanno mostrato caratteristiche tipiche spasmi motilità osservati direttamente nelle camere di flusso microfluidica, mentre mutante XfΔpliG esposto il fenotipo carente spasmi. Questo studio dimostra che pilg contribuisce alla motilità spasmi di X. fastidiosa. La camera di flusso microfluidica viene utilizzato come mezzo per osservare spasmi motilità.

La presenza di una frangia colonia periferico indicativa di tipo IV pilus-mediata spasmi motilità, è stato osservato nelle colonie di X. fastidiosa wild type e complementare ceppo Xf ΔpliG -C (Figura 1). Mutante XfΔpliG, tuttavia, non ha evidenziato una frangia intorno alla periferia delle colonie (Figura 1). Time-lapse imaging di cellule batteriche in camere a flusso nano-microfluidica ha rivelato che spasmi motil...

Watch this Scientific Journal Video about Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa at JoVE.com

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

In questo studio, una camera di flusso nano-microfluidica è stato impiegato per visualizzare e caratterizzare funzionalmente la motilità contrazione del Xylella fastidiosa, un batterio che causa la malattia di Pierce a Grapevine.

Visualizzazione di Spasmi motilità e caratterizzazione del ruolo del<em&gt; Pell</em&gt; in<em&gt; Fastidiosa Xylella</em

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility ...

The over goal of this protocol is to functionally characterize the genes responsbile for the twitching motility of bacteria using a nano microfluidic flow chamber integrated with a time-lapse image recording system.This method can help answer key questions about a bacteria phenotype and associated gene function, such as the twitching motility of bacteria.The main advantage of this technique is that it provides a special and a tempered observation for aggregation migration of individual cells and populations.Demonstrating this procedure will be Dr.Xiangyang Shi, a post-doc from my laboratory.Using a standardized deletion strategy, grow Temecula wild-type Xylella fastidiosa, grow pilG deletion mutants, and grow the complimentary strain all on PD2 medium agar plates.Incubate the bacteria at 28

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Research

JoVE Journal

Immunology and Infection

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

10.6K Views.  University of California, Davis. The over goal of this protocol is to functionally characterize the genes responsbile for the twitching motility of bacteria using a nano microfluidic flow chamber integrated with a time-lapse image recording system.This method can help answer key questions about a bacteria phenotype and associated gene function, such as the twitching motility of bacteria.The main advantage of this technique is that it provides a special and a tempered observation for aggregation migration of individual cells ...

Video: Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-&alpha; knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.

Esaminando il ruolo della rinofaringeo associata Tissue linforeticolare (NALT) nelle risposte mouse ai vaccini

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

Ricerca

Immunologia e infezioni

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Una valutazione quantitativa della migrazione cellulare con il Test Phagokinetic motilità Traccia

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Saggi qualitativi e quantitativi per il rilevamento e la caratterizzazione di proteine ​​antimicrobici

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Un metodo rapido per la lavorazione del sangue per aumentare la resa dei livelli plasmatici del peptide nel sangue umano

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

Saggi per studiare il ruolo di Vitronectin nell'adesione batterica e sulla resistenza del siero

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Isolamento, caratterizzazione e purificazione dei macrofagi dai tessuti colpiti da infiammazione obesità

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) containing immunologically active molecules. Knowledge regarding the molecular mechanisms of vesicle biogenesis, the content of the vesicles, and their functions at the pathogen-host interface is very limited. Addressing these questions requires rigorous procedures for isolation, purification, and validation of EVs. Previously, vesicle production was found to be enhanced when M. tuberculosis was exposed to iron restriction, a condition encountered by Mtb in the host environment. Presented here is a complete and detailed protocol to isolate and purify EVs from iron-deficient mycobacteria. Quantitative and qualitative methods are applied to validate purified EVs.

Mycobacterium tuberculosis shows increased production and release of extracellular vesicles in response to low iron conditions. This work details a protocol for generating low iron conditions and methods for the purification and characterization of mycobacterial extracellular vesicles released in response to iron deficiency.

Isolamento e caratterizzazione delle vescicoli extracellulari prodotti da micobatteri a limite di ferro

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) ...

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

Visualizzazione, quantificazione e mappatura delle popolazioni di cellule immunitarie nel microambiente tumorale

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

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.

Visualizzazione della resistenza batterica utilizzando sonde antibiotici fluorescenti

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

Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be altered by severe brain injury. This is evidenced by the fact that pneumonia is a major cause of mortality in patients who have suffered from ischemic stroke. The goal of this protocol is to describe the use of multicolor flow cytometric analysis to identify 13 types of immune cells in the lungs of mice, including alveolar macrophages, interstitial macrophages, CD103+ or CD11b+ dendritic cells (DCs), plasmacytoid DCs, eosinophils, monocytes/monocyte-derived cells, neutrophils, lymphoid-derived&#160; T and B cells, NK cells, and NKT cells, following ischemic stroke induction by transient middle cerebral artery occlusion. Moreover, we describe the preparation of lung homogenates using a bead homogenization method, to determine the expression levels of 13 different cytokines or chemokines simultaneously by multiplex bead arrays coupled with flow cytometric analysis. This protocol can also be used to investigate the pulmonary immune response in other disease settings, such as infectious lung disease or allergic disease.

This protocol describes the use of flow cytometry to identify the changes in immune cell composition, cytokine profile, and chemokine profile in the pulmonary environment following transient middle cerebral artery occlusion, a murine model of ischemic stroke.

Caratterizzazione di cellule immunitarie e mediatori proinfiammatori in ambiente polmonare

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be ...