This work was supported by ERC starting grants (ERC-2011-StG 280255 and ERC-2013-StG 337479) and by the Netherlands Organization for Health Research and Development (Vidi 91713319).

<ol>
	<li>Aoki, K., Kamioka, Y., Matsuda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+resonance+energy+transfer+imaging+of+cell+signaling+from+in+vitro+to+in+vivo:+basis+of+biosensor+construction,+live+imaging,+and+image+processing.">Fluorescence resonance energy transfer imaging of cell signaling from in vitro to in vivo: basis of biosensor construction, live imaging, and image processing.</a> Dev. Growth Differ. 55 (4), 515-522 (2013).</li><li>Jares-Erijman, E. A., Jovin, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRET+imaging.">FRET imaging.</a> Nature Biotechnol. 21 (11), 1387-1395 (2003).</li><li>Mank, M., 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+FRET-based+calcium+biosensor+with+fast+signal+kinetics+and+high+fluorescence+change.">A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change.</a> Biophys. J. 90 (5), 1790-1796 (2006).</li><li>Li, I. T., Pham, E., Truong, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+biosensors+based+on+the+principle+of+fluorescence+resonance+energy+transfer+for+monitoring+cellular+dynamics.">Protein biosensors based on the principle of fluorescence resonance energy transfer for monitoring cellular dynamics.</a> J. Biotechnol. Lett. 28 (24), 1971-1982 (2006).</li><li>Stephens, D. J., Allan, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+microscopy+techniques+for+live+cell+imaging.">Light microscopy techniques for live cell imaging.</a> Science. 300 (5616), 82-86 (2003).</li><li>Merkx, M., Golynskiy, M. V., Lindenburg, L. H., Vinkenborg, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+FRET+sensor+proteins+based+on+mutually+exclusive+domain+interactions.">Rational design of FRET sensor proteins based on mutually exclusive domain interactions.</a> Biochem. Soc. Trans. 41 (5), 1201-1205 (2013).</li><li>Lindenburg, L. H., 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=Quantifying+Stickiness:+Thermodynamic+Characterization+of+Intramolecular+Domain+Interactions+To+Guide+the+Design+of+Fo&#776;rster+Resonance+Energy+Transfer+Sensors.">Quantifying Stickiness: Thermodynamic Characterization of Intramolecular Domain Interactions To Guide the Design of Fo&#776;rster Resonance Energy Transfer Sensors.</a> Biochem. 53 (40), 6370-6381 (2014).</li><li>van der Velden, L. M., 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=Monitoring+bile+acid+transport+in+single+living+cells+using+a+genetically+encoded+F&#246;rster+resonance+energy+transfer+sensor.">Monitoring bile acid transport in single living cells using a genetically encoded F&#246;rster resonance energy transfer sensor.</a> J. Hepatol. 57 (2), 740-752 (2013).</li><li>Dawson, P. 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=The+heteromeric+organic+solute+transporter+&#945;-&#946;,+Ost&#945;-Ost&#946;,+is+an+ileal+basolateral+bile+acid+transporter.">The heteromeric organic solute transporter &#945;-&#946;, Ost&#945;-Ost&#946;, is an ileal basolateral bile acid transporter.</a> J. Biol. Chem. 280 (8), 6960-6968 (2005).</li><li>Meier, P. J., Stieger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bile+salt+transporters.">Bile salt transporters.</a> Annu. Rev. Physiol. 64 (1), 635-661 (2002).</li><li>Klarenbeek, J. B., Goedhart, J., Hink, M. A., Gadella, T. W., Jalink, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mTurquoise-based+cAMP+sensor+for+both+FLIM+and+ratiometric+read-out+has+improved+dynamic+range.">A mTurquoise-based cAMP sensor for both FLIM and ratiometric read-out has improved dynamic range.</a> PLoS One. 6 (4), e19170 (2011).</li><li>Wallrabe, H., Periasamy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+protein+molecules+using+FRET+and+FLIM+microscopy.">Imaging protein molecules using FRET and FLIM microscopy.</a> Curr. Opin Biotechnol. 16 (1), 19-27 (2005).</li><li>Plass, J. 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=Farnesoid+X+receptor+and+bile+salts+are+involved+in+transcriptional+regulation+of+the+gene+encoding+the+human+bile+salt+export+pump.">Farnesoid X receptor and bile salts are involved in transcriptional regulation of the gene encoding the human bile salt export pump.</a> J. Hepatol. 35 (3), 589-596 (2002).</li></ol>

The authors have nothing to disclose.

Aqui é apresentado um protocolo detalhado para a utilização de um sensor de ácido biliar romance geneticamente codificado capaz de controlar a dinâmica espaço-temporais de transporte de ácido biliar em células vivas. Este biossensor consiste em proteínas fluorescentes cerúleas e citrino que são fundidos para FXR-LBD, formando deste modo um sensor de ácido biliar à base de FRET (BAS). O Sensor Ácido Bile é relativamente simples e conveniente em uso quando se tem experiência b?...

Förster Ressonância Energia Transferência (FRET) é amplamente utilizado para obter uma melhor compreensão das funções celulares em células vivas com alta resolução temporal e espacial 1. Na FRET, a energia a partir de um fluoróforo dador animado é transferido para um fluoróforo aceitador. Eficiência de TERF é fortemente dependente da distância entre o fluoróforo dador e o aceitador e a sua orientação e é, por conseguinte, um visor sensível de mudanças conformacionais que afectam os dois fluoróforos. Este fenômeno é explorada para gerar biosensores baseados em FRET para a imagem de pequenas moléculas. Alterações na sua concentração pode ser monitorado à medida que aumenta / diminui na proporção de intensidade de emissão do aceitador em relação ao fluoróforo dador 2. Por exemplo, os biossensores de cálcio baseados em FRET permite a detecção rápida e estável de concentrações de cálcio livres nas células 3 viva. Outras vantagens de biosensores baseados em FRET são imagem latente em células vivas individuais, therdeiro não-invasivo, sua capacidade de ser direcionados para diferentes tipos de células e compartimentos celulares 4. Muitos aspectos da dinâmica de ácidos biliares intracelular ainda são pouco conhecidos. Por exemplo, pouco se sabe sobre o mecanismo de regulação subjacente de transporte de ácido biliar conjugado e não conjugado. Técnicas existentes para monitorar este transporte fazer uso principalmente de repórteres à base de luciferase, ácidos biliares radiomarcados, ou fluorescentes análogos de ácidos biliares. O último requer a modificação de ácidos biliares, possivelmente afetando suas propriedades. Repórteres à base de luciferase tem resolução de tempo pobre. Além disso, estas técnicas de resultar em perda da amostra e não são aplicáveis ​​para a imagem latente em células individuais. Por isso, seria benéfico utilizar métodos que permitem a imagiologia de célula única ao vivo da actividade de transporte de FRET utilizando biossensores, especialmente uma vez que inclui a vantagem de a detecção raciométrica 5, 6. Embora variantes de CFP / YFP forma mais frequentemente utilizada pares FRET, novas estratégias usando Morange e mCherry transportando mutações auto-associação indutores levaram a uma expansão da caixa de ferramentas FRET com novos sensores, incluindo um sensor de ácido biliar vermelho-deslocada 7. Nós previamente criado um FRET do sensor geneticamente codificado ácido biliar (BAS), que consiste de um fluoróforo dador (azul celeste) e um fluoróforo aceitador (citrino) que estão fundidos com o receptor X farnesoid (FXR) ligando de domínio de ligação (FXR-LBD) e um péptido que contém um motivo LXXLL 8. Este péptido associados com o FXR-LBD de um modo dependente-ácidos biliares. Após a activação FXR, a distância entre o citrino e azul celeste irá alterar devido a uma alteração conformacional. Em linhas celulares de mamíferos, FXR resultados da activação de um aumento claramente detectável na relação de citrino / azul celeste, enquanto o sensor purificada funciona no sentido oposto e leva a uma diminuição do rácio de FRET após activação FXR. Este sensor (CytoBAS)permite o monitoramento da dinâmica dos ácidos biliares citosólicas. Através da adição do terminal carboxilo de motivos alvo subcelulares, a construção pode ser BAS encaminhada para o núcleo (NucleoBAS) e peroxissomas (PeroxiBAS), permitindo medições de concentrações de ácidos biliares em diferentes compartimentos celulares. Embora a adição do motivo de direccionamento peroxissomal não prejudicar a sua capacidade de resposta aos ácidos biliares, células permeáveis ​​FXR-ligandos não induziu qualquer alteração de FRET PeroxiBAS dentro peroxissomas 8. Como a natureza dessa discrepância é desconhecida, o protocolo abaixo está focada em CytoBAS e NucleoBAS. A utilização deste sensor de FRET geneticamente codificado foi recentemente demonstrada em células contendo os transportadores de ácido biliar hepáticas Na + / taurocolato de co-transporte polipéptido (NTCP) e soluto orgânico alfa / beta transportador (8) OSTαβ. PNCT é o principal importador de ácido biliar hepática e OSTαβ é um bile intestinal basolateraltransportador de ácido que pode funcionar tanto como um importador e exportador dependente do gradiente de concentração de ácido biliar eletroquímica 9, 10. Dados recentes mostraram que mediante transporte de ácido biliar por NTCP e / ou OSTαβ, respostas rápidas e robustas em relação FRET como um resultado da interacção ligando-FXR-LBD pode ser observada. Aqui, descrevemos protocolos detalhados para métodos para medir FRET como a análise microscópica confocal e de células activada por fluorescência (FACS), realce passos críticos, face a eventuais problemas e discutir métodos alternativos. Usando este sensor de FRET geneticamente codificado, interacção de ácidos biliares com FXR-LBD pode ser quantificado e monitorada directamente em células vivas e proporciona um método rápido e simples de visualizar transporte de ácido biliar e dinâmica em tempo real. Plasmídeos de expressão de mamífero que codifica CytoBAS NucleoBAS e estão comercialmente disponíveis. Portanto, esse biosensor pode contribuir ainda mais para ocompreensão dos transportadores de ácidos biliares ou compostos que ativam FXR e fornecem uma visão mais profunda da biologia ácido biliar e sinalização.

<table border="0" cellpadding="0" cellspacing="0" width="867">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">CytoBAS&#160;</td>
			<td style="width:173px;">Addgene</td>
			<td align="right" style="width:145px;">62860</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NucleoBAS&#160;</td>
			<td style="width:173px;">Addgene</td>
			<td align="right">62861</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Dulbecco&#39;s modified Eagles media (DMEM)</td>
			<td style="width:173px;">Lonza</td>
			<td>BE12-614F</td>
			<td>High glucose without L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Penicillin-Streptomycin (pen/strep)</td>
			<td style="width:173px;">Lonza</td>
			<td style="width:145px;">17-602E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">L-glutamine (200mM)</td>
			<td style="width:173px;">Lonza</td>
			<td style="width:145px;">17-605E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:173px;">Invitrogen</td>
			<td style="width:145px;">102-70</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Trypsin-EDTA (10x)</td>
			<td style="width:173px;">Lonza</td>
			<td style="width:145px;">CC-5012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">T-25 cell culture flask</td>
			<td style="width:173px;">VWR international</td>
			<td style="width:145px;">392-0253</td>
			<td>Laminin coated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">T-175 cell culture flask</td>
			<td style="width:173px;">VWR international</td>
			<td style="width:145px;">392-0238</td>
			<td>Laminin coated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">6-well plate</td>
			<td style="width:173px;">VWR international</td>
			<td style="width:145px;">734-0229</td>
			<td>Poly-L-lysine and Laminin coated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">10cm dish</td>
			<td style="width:173px;">VWR international</td>
			<td style="width:145px;">392-0243</td>
			<td>Laminin coated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Diethylaminoethyl (DEAE) - Dextran</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:145px;">D9885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Polyethylenimine (PEI)&#160;</td>
			<td style="width:173px;">Brunschwig</td>
			<td style="width:145px;">23966-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">G418 (geneticin) 50 mg/ml</td>
			<td style="width:173px;">Invitrogen</td>
			<td style="width:145px;">10131-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Hygromycin B, 50 mg / ml</td>
			<td style="width:173px;">Invitrogen</td>
			<td style="width:145px;">10687-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Cloning cylinder (6x8 mm)</td>
			<td style="width:173px;">Bellco</td>
			<td style="width:145px;">2090-00608</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">L-15 Leibovitz culture medium</td>
			<td style="width:173px;">Invitrogen</td>
			<td style="width:145px;">21083-027</td>
			<td>No phenol red</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:300px;">Polystyrene round bottom tube (5 ml) Facs tube</td>
			<td style="width:173px;">Falcon BD</td>
			<td style="width:145px;">352008</td>
			<td>No cap, non-sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Falcon 2063 tubes (5 ml)</td>
			<td style="width:173px;">Falcon BD</td>
			<td align="right" style="width:145px;">352063</td>
			<td>Snap cap, sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Nunc Lab-Tek 8 well coverglass</td>
			<td style="width:173px;">Thermo scientific</td>
			<td style="width:145px;">155409</td>
			<td>Sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Charcoal-filtered FBS</td>
			<td style="width:173px;">Life technologies</td>
			<td style="width:145px;">12676011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">GW4064</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:145px;">G5172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">TCDCA</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:145px;">T6260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">CDCA</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:145px;">C9377</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Other chemicals</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:145px;">n.v.t.</td>
			<td></td>
		</tr>
	</tbody>
</table>

real time monitoring of intracellular bile acid dynamics using a genetically encoded fret-based bile acid sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

O sensor de FRET-BAS apresentada baseia-se no domínio de FXR (LBD-FXR) de ligação ao ligando ligado a dois fluoróforos de citrino e cerúleas) e um motivo LXXLL. Este sensor permite investigações de transporte de ácido biliar em células com elevada resolução espacial e temporal (Figura 1 A) vivas. Mutações em celeste e citrino foram aplicados para promover a formação do complexo intramolecular (Figura <stro...

Watch this Scientific Journal Video about Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor at JoVE.com

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

Monitoramento em Tempo Real de intracelular Bile Ácido Dynamics usando um sensor Ácido Bile baseado em FRET codificados geneticamente

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

real-time-monitoring-intracellular-bile-acid-dynamics-using

Research

JoVE Journal

Bioengineering

9.8K visualizações.  Academic Medical Center. We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types. 

Vídeo: Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

Sensor de microagulhas baseado oco para Multiplexed Transdermal Eletroquímica Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Bioengenharia

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

Citometria de massa: Protocolo para Ajuste Diário e amostras de células em execução em um citômetro de Massa CyTOF

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Glutamine Flux Imaging Using Genetically Encoded Sensors

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories.
One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.

This article will demonstrate how to monitor glutamine dynamics in live cells using FRET. Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed for genetically encoded glutamine sensors.

Glutamina Flux imagem usando sensores codificados geneticamente

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3D Orbital Rastreamento em um microscópio de dois fotões Modificação: Uma Aplicação para o rastreamento de vesículas intracelulares

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

Monitoramento em tempo real de High Intensity Focused Ultrasound (HIFU) Ablação de<em&gt; In Vitro</em&gt; Canine Fígados Usando Movimento Harmônico Imaging para ultra-som focalizado (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

Medição de TCR-pMHC Binding<em&gt; In Situ</em&gt; Usando a microscopia Ensaio de FRET

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

Sustained Administration of &#946;-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic &#946;-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to &#946;-cells in culture and in transplanted tissue models. These technologies allow the study of candidate &#946;-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating &#946;-cell proliferation in a culture system is critical before moving forward to in vivo models. Herein, we describe a method to co-culture intact mouse islets with biodegradable compound of interest (COI)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for the purpose of assessing the effects of sustained in situ release of mitogenic factors on &#946;-cell proliferation. This technique describes in detail how to generate PLGA microspheres containing a desired cargo using commercially available reagents. While the described technique uses recombinant human Connective tissue growth factor (rhCTGF) as an example, a wide variety of COI could readily be used. Additionally, this method utilizes 96-well plates to minimize the amount of reagents necessary to assess &#946;-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of &#946;-cell proliferation. This method is suitable for determining the efficacy of candidate &#946;-cell mitogens.

Administração sustentada dos mitógenos de células β para intactas mouse Ilhotas<em&gt; Ex Vivo</em&gt; Usando biodegradável poli (ácido láctico-co-glicólico) Microesferas

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the ...

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing termini of oligo- and polysaccharides, as well as its unique chemical characteristics, sialic acid is involved in a multitude of different receptor-ligand interactions. By modifying the expression of sialic acid on the cell surface, sialic acid-dependent interactions will consequently be influenced. This can be helpful to investigate sialic acid-dependent interactions and has the potential to influence certain diseases in a beneficial way. Via metabolic glycoengineering (MGE), the expression of sialic acid on the cell surface can be modulated. Herein, cells, tissues, or even entire animals are treated with C2-modified derivatives of N-acetylmannosamine (ManNAc). These amino sugars act as sialic acid precursor molecules and therefore are metabolized to the corresponding sialic acid species and expressed on glycoconjugates. Applying this method produces intriguing effects on various biological processes. For example, it can drastically reduce&#160;the expression of polysialic acid (polySia) in treated neuronal cells and thus affects neuronal growth and differentiation. Here, we show the chemical synthesis of two of the most common C2-modified N-acylmannosamine derivatives, N-propionylmannosamine (ManNProp) as well as N-butanoylmannosamine (ManNBut), and further show how these non-natural amino sugars can be applied in cell culture experiments. The expression of modified sialic acid species is quantified by high performance liquid chromatography (HPLC) and further analyzed via mass spectrometry. The effects on polysialic acid expression are elucidated via Western blot using a commercially available polysialic acid antibody.

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid is a typical monosaccharide-unit found in glycoconjugates. It is involved in a plethora of molecular and cellular interactions. Here we present a method to modify cell surface sialic acid expression using metabolic glycoengineering with N-acetylmannosamine derivatives.

Glycoengineering metabólica do ácido siálico usando N-acil-modificado Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Um protocolo para rastreamento em tempo real 3D única partícula

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Preparação, purificação e utilização de lipossomas contendo ácido graxo

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...