This research was supported by the research programs of the National Research Foundation of Korea (NRF) (NRF-2015R1A1A1A05001548 and NRF-2015R1A5A1037668).

Esperimenti con campioni di sangue sono stati eseguiti in conformità con le leggi e le linee guida istituzionali. I campioni sono stati i campioni di sangue residui che erano stati acquisiti ed elaborati in test clinici presso l&#39;istituto. 1. PT-AS Setup ottico NOTA: Si può utilizzare un tubo vuoto micro-capillare per una configurazione iniziale PT-AS. <ol><li> Montare un tubo micro-capillare vuota con diametri interno ed esterno rispettivamente di 200 e 330 micron, e una lunghezza superiore a ~ 5 cm un dispositivo capillare. infissi fibre disponibili in commercio possono essere usati come proiettori tubo. </li><li> Trovare ancorare un puntatore laser 650 nm, cioè, sondare sorgente luminosa, per illuminare il tubo capillare. Il fascio sonda deve essere maggiore del tubo capillare. Posizionare uno schermo (ad esempio, carta bianca) dietro il tubo capillare per osservare un reticolo periodico angolare. </li><li> Per la parte di rilevamento, rimuovere le lenti in una webcam per catturare direttamente il Scattrendo modello. Posizionare la webcam dietro il tubo capillare con un angolo di 25-35 ° rispetto alla direzione del fascio sonda. Assicurarsi che il modello periodica angolare prodotta dal tubo capillare può essere misurata con il rivelatore (Figura 1). Osservare il modello periodica angolare nel mezzo del sensore di immagine quando il sensore è posizionato correttamente. </li><li> Posizionare una sorgente di luce di eccitazione PT 532 nm per illuminare il tubo capillare. Posizionare la sorgente luminosa PT con qualsiasi angolazione, finché la luce di eccitazione PT sovrappone con fascio sonda sul tubo capillare e non raggiunge direttamente il rivelatore. PT eccitazione dei campioni di sangue utilizzando alta potenza ottica tipicamente migliora la PT-AS sensibilità, in quanto porta ad un cambiamento maggiore nella RI. <ol><li> Utilizzare la massima potenza ottica della sorgente di luce di eccitazione PT impiegato. Inoltre, assicurarsi che la luce di eccitazione PT si sovrappone alla luce sonda sul tubo capillare. Utilizzare una dimensione del fascio di luce di eccitazione PTalmeno doppia di quella della luce sonda per riscaldare l&#39;intero volume della sonda. </li></ol></li><li> Collocare un filtro passa lungo davanti al rivelatore per bloccare la luce 532 nm e misurare solo la luce della sonda 650 nm. </li><li> Installare un chopper nel cammino ottico della luce di eccitazione PT prima illuminare il tubo capillare. Il chopper ottico è utilizzato per modulare l&#39;intensità PT eccitazione luminosa. </li></ol> 2. Preparazione del campione di sangue <ol><li> Disegnare 6 ml di sangue intero fresco in condizione anemica in tubi di campionamento del sangue acido etilendiamminotetraacetico, e mescolare bene i campioni. Non è richiesta alcuna altra elaborazione. </li><li> Misurare i campioni di sangue utilizzando il sensore PT-AS entro 24 ore di estrazione per prevenire la coagulazione. </li></ol> 3. PT-AS Protocolli di misura <ol><li> Caricare un tubo micro-capillare con un campione di sangue per misurare. Riempire il capillare del sangue attraverso l&#39;azione capillare inserendo il tubo nel sangue sampio. Il volume di campione minimo richiesto per la misurazione è determinata dal diametro interno del tubo capillare e la dimensione fascio sonda. <ol><li> Impiegare un tubo con un diametro interno di 200 micron. La dimensione fascio sonda era di 2 mm nei risultati rappresentativi, suggerendo che la misurazione può essere eseguita con un volume di campione di&gt; 63 nl. </li></ol></li><li> Montare il tubo capillare nella posizione indicata nel dispositivo. </li><li> Accendere il laser della sonda 650 nm per illuminare il tubo micro-capillare del sangue-caricato. Il modello periodico angolare deve essere osservato con la webcam. </li><li> Accendere il 532-nm laser PT di eccitazione per illuminare il tubo. </li><li> Eseguire il chopper ottico per modulare l&#39;intensità della luce di eccitazione PT a 2 Hz. NOTA: Il razionale per la selezione di questa condizione di funzionamento è descritto in discussione e Kim et al. 12. <ol><li> Montare una ruota chopper nel montaggio testa del motore del chopper otticasistema. </li><li> Selezionare la casella di controllo del chopper, e utilizzare la manopola di comando nella console per impostare la frequenza di modulazione. </li><li> Eseguire l&#39;elicottero con la manopola di controllo. </li></ol></li><li> Registrare il modello di diffusione fluttuante tramite la webcam per 5 secondi in formato MPEG-4 (mp4). </li></ol> 4. Signal Processing NOTA: l&#39;elaborazione del segnale PT-AS è stata effettuata utilizzando un codice MATLAB laboratorio sviluppato. <ol><li> Caricare il file video per estrarre le immagini. Per ogni immagine [vedere figura 2 (a) per una immagine rappresentativa], ottenere il pattern dispersione media calcolando la media dei valori dei pixel lungo la direzione verticale [Figura 2 (b, c)]. </li><li> Valutare la trasformata di Fourier del modello dispersione media, e calcolare la fase alla frequenza spaziale picco. Eseguite le operazioni per tutti i fotogrammi di tutte le immagini registrate. </li><li> Utilizzando i valori di fase ottenuti da tutte le immagini, tracciare la fase temporalefluttuazione [Figura 2 (d)]. Si noti che la fase oscilla alla frequenza di modulazione PT. Prendere la trasformata di Fourier della fluttuazione di fase nel dominio del tempo, e di ottenere l&#39;ampiezza alla frequenza di modulazione. Questo segnale è indicato come PT-AS segnale [figura 2 (e)]. </li><li> Misurare la [Hb] di un campione di sangue convertendo il suo segnale PT-AS nella corrispondente [Hb] alla curva di taratura che si ottiene nel protocollo 5. </li></ol> 5. PT-AS Calibrazione <ol><li> Preparare campioni di sangue, con [Hb] valori che sono uniformemente distribuiti nel campo di rilevamento del sensore PT-AS (ad esempio 0 - 18 g / dl). </li><li> Prima di calibrazione, quantificare i valori [Hb] dei campioni utilizzando un analizzatore ematologico di riferimento. Misurare i segnali PT-AS dei campioni. </li><li> Ricavare una curva di calibrazione relativa [Hb] al segnale PT-AS eseguendo un lineare minimi quadrati, [Hb] = A [PT-AS segnale] + B, delle experimental risultati. Per le condizioni di esercizio di cui alla tabella 1, il rapporto tra [Hb] e il segnale di PT-AS è risultato essere [Hb] = 5.13 [segnale PT-AS] - 0.09. Usa il codice MATLAB per eseguire la misura lineare. </li></ol>

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No conflict of interest is declared.

Il sensore PT-AS rappresenta un metodo completamente ottica in grado di misurare direttamente [Hb] di campioni di sangue non trasformati. Il metodo quantifica [Hb] nel sangue utilizzando la risposta PT intrinseca di molecole di emoglobina negli eritrociti. Sotto illuminazione da luce 532 nm, le molecole di Hb assorbono l&#39;energia luminosa e producono calore. L&#39;aumento di temperatura risultante cambia il RI del campione di sangue. L&#39;elevata sensibilità del RI di BSI è stata sfruttata per misurare questo camb...

Un esame del sangue è comunemente eseguita per valutare la salute umana in generale e per rilevare i biomarcatori legati a determinate malattie. Ad esempio, la concentrazione di colesterolo nel sangue serve come criterio per iperlipidemia, che è strettamente correlato a malattie cardiovascolari e pancreatite. Il contenuto di glucosio nel sangue devono essere misurati di frequente, come il livello di glucosio è associata a complicazioni come la chetoacidosi diabetica e la sindrome iperosmolare iperglicemici. gravi malattie come la malaria, il virus dell&#39;immunodeficienza umana e la sindrome da immunodeficienza acquisita sono diagnosticati da esami del sangue, e la quantificazione dei componenti del sangue, tra cui eritrociti, trombociti, leucociti e consente lo screening del pancreas e malattie renali. L&#39;emoglobina (Hb), una componente fondamentale del sangue, rappresenta circa il 96% degli eritrociti, e trasporta l&#39;ossigeno a organi umani. alterazione significativa della sua concentrazione di massa ([Hb]) può indicare memodifiche tabolic, malattie epatobiliari e neurologiche, cardiovascolari e disturbi endocrinologici 1. [Hb] è quindi regolarmente misurato in esami del sangue. In particolare, i pazienti anemici, pazienti in dialisi, e le donne in gravidanza sono fortemente raccomandati per il monitoraggio [Hb] come un compito di vitale importanza 2. Sono stati così sviluppati diversi [Hb] metodi di rilevamento. Il metodo emoglobina cianuro, una delle tecniche più comuni per [Hb] quantificazione, impiega cianuro di potassio (KCN) per distruggere il doppio strato lipidico di eritrociti 3. L&#39;emoglobina cianuro prodotto dalla esposizioni chimiche ad alto assorbimento intorno a 540 nm; quindi, [Hb] misurazioni possono essere effettuate tramite analisi colorimetrica. Questo metodo è ampiamente utilizzato per la sua semplicità, ma i prodotti chimici utilizzati (ad esempio, KCN e ossido dimethyllaurylamine) sono tossici per l&#39;uomo e per l&#39;ambiente. Il regime ematocrito misura il rapporto del volume dei globuli rossi rispetto al totale vol sangueume attraverso separazione centrifuga; tuttavia richiede un volume relativamente grande di sangue (50-100 ml) 4. Spettrofotometria metodi di misura [Hb] appunto senza alcun prodotto chimico, ma misure a lunghezze d&#39;onda multiple e un grande volume di sangue sono necessari 5,6. Analogamente, sono stati proposti diversi metodi ottici di misura [Hb] compresi metodi di rilevamento basato su luce-dispersione, ma i loro precisione delle misure dipende fortemente dalla precisione del modello sangue teorico. Per superare queste limitazioni, sono stati recentemente proposti [Hb] metodi di rilevamento basati sull&#39;effetto fototermico (PT) di Hb 7. Hb, che è composto principalmente da ossidi di ferro, assorbe la luce a 532 nm e converte l&#39;energia luminosa in calore 8-10. Questo aumento di temperatura PT può essere rilevato otticamente misurando una variazione dell&#39;indice di rifrazione (RI) dei campioni di sangue. Yim et al. impiegato spettrale-dominio di coerenza ottica reflectometry per misurare la variazione ottica percorso di lunghezza PT in una camera di sangue contenente 11. Sebbene il metodo consente chemical free e misurazione diretta [Hb], l&#39;uso di uno spettrometro e una disposizione interferometrica possono ostacolare la sua miniaturizzazione. Recentemente abbiamo presentato un metodo di rivelazione [Hb], chiamato sensore foto-termico angolare light scattering (PT-AS), che è più adatto per il dispositivo di miniaturizzazione 12. Il sensore PT-AS sfrutta l&#39;elevata sensibilità RI dell&#39;interferometria retrodiffusione (BSI) per misurare le variazioni nel PT RI di un campione di sangue all&#39;interno di un tubo capillare. BSI sono stati utilizzati per misurare RI di varie soluzioni 13-15 e per monitorare le interazioni biochimiche in soluzione free 16. Il sensore PT-AS impiega disposizione ottica simile come in BSI, ma combina l&#39;installazione di eccitazione fototermico per misurare aumento PT di RI in campioni di sangue. principi di funzionamento del BSI e sensori PT-AS sono descritti in dettaglio altrove <sup&gt; 12,15. PT-AS sensore dimostrato alta sensibilità di misura [Hb] in un ampio campo di rilevamento (0,35-17,9 g / dl) ed è in grado di funzionare con volumi di campione di &lt;100 nl. Non è richiesto alcun condizionamento del campione di sangue, e il tempo di misurazione è solo ~ 5 sec. Qui, l&#39;apparato sperimentale e una dettagliata protocollo di misurazione sono descritti. Risultati rappresentativa PT-AS sono forniti utilizzando campioni di sangue di pazienti anemici, ed i risultati vengono confrontati con quelli di un analizzatore ematologico per valutare la precisione del sensore PT-AS.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>650nm laser pointer</td>
			<td>LASMAC</td>
			<td>LED-1</td>
			<td>Probe light</td>
		</tr>
		<tr>
			<td>Hollow round glass capillaries</td>
			<td>Cm Scientific</td>
			<td>CV2033</td>
			<td>Blood sample container</td>
		</tr>
		<tr>
			<td>Webcam</td>
			<td>Logitech</td>
			<td>C525</td>
			<td>CMOS optical sensor</td>
		</tr>
		<tr>
			<td>532-nm DPSS laser</td>
			<td>CNI Laser</td>
			<td>MGL-&#8546;-532</td>
			<td>Photothermal light source</td>
		</tr>
		<tr>
			<td>Optical chopper system</td>
			<td>Thorlabs</td>
			<td>MC2000-EC</td>
			<td>Optical chopper</td>
		</tr>
		<tr>
			<td>Plastic long-pass filter</td>
			<td>Edmund Optics</td>
			<td>#43-942</td>
			<td>To reject 532-nm PT excitation light</td>
		</tr>
		<tr>
			<td>Fiber clamp</td>
			<td>Thorlabs</td>
			<td>SM1F1-250</td>
			<td>Capillary tube fixture</td>
		</tr>
		<tr>
			<td>EDTA coated blood sampling tube</td>
			<td>Greiner Bio-One</td>
			<td>VACUETTE 454217</td>
			<td>Blood sampling &#38; anticoagulating</td>
		</tr>
		<tr>
			<td>Hematology analyzer</td>
			<td>Siemens AG</td>
			<td>ADVIA 2120i</td>
			<td>Reference hematology analyzer</td>
		</tr>
	</tbody>
</table>

a rapid and chemical-free hemoglobin assay with photothermal angular light scattering

Photo-termica angolare light scattering (PT-AS) è un metodo ottico innovativo per la misurazione della concentrazione di emoglobina ([Hb]) dei campioni di sangue. Sulla base della risposta fototermico intrinseca delle molecole di emoglobina, il sensore permette ad alta sensibilità, misura privo di sostanze chimiche di [Hb]. [Hb] capacità di rilevazione con un limite di 0,12 g / dl nel range di 0,35 - 17.9 g / dl è stata dimostrata in precedenza. Il metodo può essere facilmente implementato utilizzando dispositivi elettronici economico consumo come un puntatore laser e una webcam. L&#39;uso di un tubo micro-capillare come un contenitore di sangue permette anche il dosaggio dell&#39;emoglobina con un volume di sangue nanolitro scala ed un basso costo di esercizio. Qui, istruzioni dettagliate per le procedure di elaborazione di impostazione PT-AS ottica e di segnale sono presentati. protocolli sperimentali e risultati rappresentativi per i campioni di sangue in condizioni anemici ([Hb] = 5,3, 7,5, e 9,9 g / dl) sono anche previste, e le misurazioni vengono confrontati con quelli froma analizzatore ematologico. La sua semplicità di implementazione e gestione dovrebbe consentire la sua ampia adozione nei laboratori clinici e di risorse limitate.

Un test dell&#39;emoglobina è stata eseguita utilizzando il sensore PT-AS, e le sue misure sono stati confrontati con quelli di un analizzatore ematologico. L&#39;esperimento è stato condotto con una intensità luminosa PT di eccitazione di 1,4 W / cm 2, PT frequenza di modulazione di 2 Hz, e misura del tempo di 5 sec. La tabella 1 riassume le condizioni sperimentali. Le dimensioni del fascio di luce di eccitazione sonda e PT erano 5,5 e 2 mm, rispettivament...

Watch this Scientific Journal Video about A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering at JoVE.com

A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

Una rapida e chimico-libera emoglobina Assay con Photothermal angolare Light Scattering

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis ...

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Bioengineering

9.6K Views.  Yonsei University. A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

Video: A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

Rapid Patterning sottrattiva di strati cellulare dal vivo con una sonda Microfluidic

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

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Bioingegneria

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formazione Light-mediata ed il modello di idrogel per colture cellulari

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Rapid Mix Preparazione di bioispirati Nanoscale idrossiapatite per applicazioni biomediche

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

Costruzione e messa a punto di un biotestattore fotosintetico di alghe a bilanciere a banco con monitoraggio della temperatura, della luce e del pH per test di crescita cinetica

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

Protocollo per il trasferimento di MicroRNA in adulto cellule staminali emopoietiche derivate da midollo osseo per abilitare ingegneria cellulare combinata con il Targeting magnetico

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria can undergo changes in the physiology due to the exposure to host growth environment and the presence of the host cells. We developed an assay to rapidly measure the virulence state of bacteria that minimize the extent to which bacteria grow in the presence of host cells. Bacteria and amoebae are mixed together and immobilized on a single imaging plane using an agar pad. The procedure uses single-cell fluorescence imaging with calcein-acetoxymethyl ester (calcein-AM) as an indicator of host cell health. The fluorescence of host cells is analyzed after 1 h of exposure of host cells to bacteria using epifluorescence microscopy. Image analysis software is used to compute a host killing index. This method has been used to measure virulence within planktonic and surface-attached Pseudomonas aeruginosa sub-populations during the initial stage of biofilm formation and may be adapted to other bacteria and other stages of biofilm growth. This protocol provides a rapid and robust method of measuring virulence and avoids many of the complexities associated with the growth and maintenance of mammalian cell lines. Virulence phenotypes measured here using amoebae have also been validated using mouse macrophages. In particular, this assay was used to establish that surface attachment upregulates virulence in P. aeruginosa.

Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.

Un'analisi di virulenza batterica basata su immagine rapida usando ameba

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria ...

Rapid Characterization of Genetic Parts with Cell-Free Systems

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing. Here, we show a rapid and cost-effective method for characterizing genetic parts. Our method utilizes cell-free lysate, prepared in-house as a medium to evaluate parts via the expression of a reporter protein. Template DNA is prepared by PCR amplification using inexpensive primers to add variant parts to the reporter gene, and the template is added to the reaction as linear DNA without cloning. Parts that can be added in this way include promoters, operators, ribosome binding sites, insulators, and terminators. This approach, combined with the incorporation of an acoustic liquid handler and 384-well plates, allows the user to carry out high-throughput evaluations of genetic parts in a single day. By comparison, cell-based screening approaches require time-consuming cloning and have longer testing times due to overnight culture and culture density normalization steps. Further, working in cell-free lysate allows the user to exact tighter control over the expression conditions through the addition of exogenous components and DNA at precise concentrations. Results obtained from cell-free screening can be used directly in applications of cell-free systems or, in some cases, as a way to predict function in whole cells.

Well-characterized genetic parts are necessary for the design of novel genetic circuits. Here we describe a cost-effective, high-throughput method for rapidly characterizing genetic parts. Our method reduces cost and time by combining cell-free lysates, linear DNA to avoid cloning, and acoustic liquid handling to increase throughput and reduce reaction volumes.

Caratterizzazione rapida di parti genetiche con sistemi cell-free

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

Fermentazioni a controllo leggero per la produzione chimica microbica e proteica

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

In vivo Imaging di tessuti biologici con fluorescenza combinata a due fotoni e microscopia a dispersione Raman stimolata

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

A Nonsequencing Approach for the Rapid Detection of RNA Editing

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger sequencing and RNA sequencing techniques. However, these methods can be costly and time-consuming. In this protocol, a portable microtemperature gradient gel electrophoresis (&#181;TGGE) system is used as a nonsequencing approach for the rapid detection of RNA editing. The process is based on the principle of electrophoresis, which uses high temperatures to denature nucleic acid samples as they move across a polyacrylamide gel. Across a range of temperatures, a DNA fragment forms a gradient of fully double-stranded DNA to partially separated strands and then to entirely separated single-stranded DNA. RNA-edited sites with distinct nucleotide bases produce different melting profiles in &#181;TGGE analyses. We used the &#181;TGGE-based approach to characterize the differences between the melting profiles of four edited RNA fragments and their corresponding nonedited (wild-type) fragments. Pattern Similarity Scores (PaSSs) were calculated by comparing the band patterns produced by the edited and nonedited RNAs and were used to assess the reproducibility of the method. Overall, the platform described here enables the detection of even single base mutations in RNAs in a straightforward, simple, and cost-effective manner. It is anticipated that this analysis tool will aid new molecular biology findings.

Rapid detection and reliable quantification of RNA editing events at a genomic scale remain challenging and currently rely on direct RNA sequencing methods. The protocol described here uses microtemperature gradient gel electrophoresis (&#181;TGGE) as a simple, quick, and portable method of detecting RNA editing.

Un approccio non sequenziale per il rilevamento rapido dell'editing dell'RNA

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger ...