Questo lavoro è stato sostenuto dalle concessioni dal Clifford signora Vecchio Bianco Graham Dotato Fondo di ricerca e il NIH (AR059838) a CB, dal NIH (AR052791, AR044387-ARRA, e NS063298) a LTT, e dal Baylor College of Medicine Citometria Ordinamento e Cell Core con finanziamenti del NIH (NCRR S10RR024574, NIAID AI036211, e NCI P30CA125123). I contenuti sono di esclusiva responsabilità degli autori e non rappresentano necessariamente il punto di vista ufficiale del NIH.

1. La colorazione delle cellule <ol><li> Preparare le cellule. Le cellule devono essere utilizzati, pur in crescita attiva (60-80 confluenza%). Siero affamare le cellule durante la notte, la piastra loro celle a 0,1 milioni di euro oltre a 0,5 ml di siero-free medio nell&#39;arco di 24 pozzetti e posto nell&#39;incubatrice (37 ° C, 5% CO 2) per 30 minuti - 2 ore per il recupero da tripsinizzazione. </li><li> Mescolare gli anticorpi primari e secondari. Mix (5 ml di primaria anticorpi anti-GLUT4 con 1 ml di pollo secondario di capra anti-IgG coniugato con AlexaFluor 488) x numero di pozzi. Incubare per 10 minuti a temperatura ambiente, al buio. </li><li> Preparare uno stock 2X lavoro di insulina da diluire in mezzo. </li><li> Verificare se le cellule hanno aderito o meno. </li><li> Mescolare delicatamente aggiungere 6 microlitri di anticorpi e 0,5 ml di vostro stock 2X lavoro di insulina per i pozzetti contenenti le cellule. Evitare qualsiasi turbolenza del mezzo. Incubare (37 ° C, 5% CO 2) per 30 minuti, al buio. </li><li> Fissare le cellule con l&#39;aggiunta di 0,5 ml di PBS + 1% PFA ad ogni bene, senza tremare le cellule. Incubare per 20 minuti a temperatura ambiente, al buio. </li><li> Se le cellule hanno aderito ai pozzi, delicatamente ascensore con un sollevatore di cellulare. Trasferire le cellule (1 totale ml) in provette che stare in una citometro a flusso e centrifugare per agglomerare le cellule. </li><li> Lavare il pellet due volte con 1 ml di PBS e risospendere in 0,4 ml di PBS + 1% PFA. A questo punto, le cellule possono essere avvolti in pellicola e conservato in frigorifero (4-8 ° C) o adottate per il citofluorimetro per l&#39;immediata acquisizione dei dati. </li></ol> 2. Acquisizione Dati <ol><li> Impostare un cancello largo intorno alle cellule vivono nel side scatter (SSC) in avanti rispetto a dispersione (FCS) del pannello. Utilizzare un tubo negativo (cellule non trattate con insulina o cellule &quot;colorate&quot; con un controllo isotipo) per impostare il parametro FL1. Assicurarsi che il picco è del tutto nel pannello, non tagliare sul lato inferiore. </li><li> Acquisire i dati di almeno 10.000 cellule per provetta. </li></ol> 3. Analisi dei dati <ol><li> Disegnare un gate intorno alle cellule vivono nel vs SSC FSC pannello (Figura 1A). Tracciare la istogrammi di AlexaFluor 488 intensità di fluorescenza in funzione del numero di cellule vive all&#39;interno della cellula-gate (Figura 1B). È possibile sovrapporre diversi istogrammi di visualizzare le differenze, ma per la quantificazione, determinare l&#39;intensità media e mediana di fluorescenza per ogni popolazione cellulare e utilizzare questi numeri per i confronti (Figura 1C). </li><li> Normalizzare i dati al valore ottenuto in assenza di insulina, come mostrato in Figure 1C e 1D. </li></ol> 4. Rappresentante Risultati In mioblasti isolati da un individuo sano (non insulino-resistenza), l&#39;insulina induce una dose-dipendente traslocazione di GLUT4 dal citoplasma alla membrana plasmatica (figura 1, a sinistra). Al contrario, l&#39;insulina non induce traslocazione GLUT4 sulla membrana plasmatica di mioblasti isolati da un paziente con insulino-resistenza (figura 1, a destra). <img src="/files/ftp_upload/2429/2429fig1.jpg" alt="Figura 1" /> Figura 1. Esempio di un esperimento di colorazione. Mioblasti sono stati isolati da un donatore, senza insulino-resistenza (a sinistra) e da un paziente con insulino-resistenza (a destra). A, SSC (scatter laterale) rispetto FCS (forward scatter) trama con un cancello intorno alle cellule vive. B, cellule in gated A, colorate con l&#39;anticorpo contro un epitopo esterno di GLUT4, e di sinistra non stimolate (rosso) o stimolati con insulina 1 nm (blu), 10 Nm (arancione), o 100 nm (verde). Per chiarezza la figura, si limita a mostrare i dati, senza e con 100 nM di insulina per le cellule con insulino-resistenza. C, media intensità di fluorescenza (MFI) degli istogrammi mostrato in B sono stati normalizzati per la MFI delle cellule non stimolate sinistra (non insulina). D, grafici dei dati normalizzati IFM dal istogrammi mostrato in B.

<ol>
	<li>Zhao, F. Q., Keating, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+and+genomics+of+glucose+transporters.">Functional properties and genomics of glucose transporters.</a> Curr. Genomics. 8, 113-128 (2007).</li><li>Karnieli, E., Armoni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+the+insulin-responsive+glucose+transporter+GLUT4+gene:+from+physiology+to+pathology.">Transcriptional regulation of the insulin-responsive glucose transporter GLUT4 gene: from physiology to pathology.</a> Am. J. Endocrinol. Metab. 295, 38-45 (2008).</li><li>Graham, T. E., Kahn, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+alterations+of+glucose+transport+and+molecular+mechanisms+of+intertissue+communication+in+obesity+and+type+2+diabetes.">Tissue-specific alterations of glucose transport and molecular mechanisms of intertissue communication in obesity and type 2 diabetes.</a> Horm. Metab. Res. 39, 717-721 (2007).</li><li>Alter, G., Malenfant, J. M., Altfeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD107a+as+a+functional+marker+for+the+identification+of+natural+killer+cell+activity.">CD107a as a functional marker for the identification of natural killer cell activity.</a> J. Immunol. Methods. 294, 15-22 (2004).</li><li>Vermes, I., Haanen, C., Steffens-Nakken, H., Reutellingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+apoptosis.+Flow+cytometric+detection+of+phosphatidylserine+expression+on+early+apoptotic+cells+using+fluorescein+labeled+Annexin+V.">A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V.</a> J. Immunol. Methods. 184, 39-51 (1995).</li><li>Lugli, E., Roederer, M., Cossarizza, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+analysis+in+flow+cytometry:+the+future+just+started.">Data analysis in flow cytometry: the future just started.</a> Cytometry A. 77, 705-713 (2010).</li></ol>

Nessun conflitto di interessi dichiarati.

Abbiamo dimostrato una tecnica rapida per quantificare la traslocazione del GLUT4 dal citoplasma alla membrana plasmatica delle cellule mediante citometria di flusso. GLUT4 è solo transitoriamente espressa a livello della membrana plasmatica delle cellule ed è endocitosi. Dal momento che gli anticorpi legati rimanere attaccato al GLUT4, anche durante endocitosi, il segnale di fluorescenza fornisce la quantità totale di GLUT4 che è stato esposto a livello della membrana plasmatica per tutta la durata scelta di incubazione in presenza di insulina. Questa tecnica può essere applicata anche allo studio della traslocazione di altre proteine ​​da un compartimento intracellulare alla membrana plasmatica, ha fornito un buon anticorpo diretto contro un epitopo esterna della proteina di interesse è disponibile. Ad esempio, un test simile è oggi comunemente utilizzato per valutare il tasso di degranulazione di linfociti T citotossici e dei linfociti natural killer misurando la traslocazione di CD107a alla loro membrana plasmatica 4,5. La traslocazione della fosfatidilserina dal lato interno della membrana plasmatica verso il lato esterno durante l&#39;apoptosi delle cellule o necrosi può essere rilevata mediante citometria di flusso, utilizzando fluoroforo marcata con annessina V 6. Oltre rapidità del test, citometria a flusso presenta il vantaggio di permettere lo studio della traslocazione delle proteine ​​alla membrana plasmatica in popolazioni di cellule miste. Infatti, si può combinare colorazione per i marcatori sottoinsieme delle cellule e per la proteina di interesse, seguita dalla acquisizione dati su un multi-laser citofluorimetro 7. Le quantità di anticorpi di cui al paragrafo 1.2 del protocollo sono un punto di partenza, si consiglia di titolare il tuo anticorpi con le vostre cellule di interesse per determinare la concentrazione minima di anticorpi che ti dà un segnale di massima. Una buona gamma di partenza per il primario di anticorpi anti-umano GLUT4 che abbiamo usato qui sarebbe 1-10 microlitri per canna (cioè anticorpi 0,2-2 mg per provetta), ogni tubo contenente non più di 1 milione di cellule. Per gli altri anticorpi primari, fare riferimento alle istruzioni del fornitore. Normalizzazione dei dati è necessario in quanto autofluorescenza di cellule varia dal donatore al donatore e da esperimento per esperimento.

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Anti-human GLUT4 antibodies</td>
<td>&nbsp;</td>
<td>Santa Cruz Biotechnologies</td>
<td>sc-1606</td>
<td>Against an external epitope of GLUT4</td>
</tr>
<tr>
<td>Chicken anti-goat IgG conjugated to AlexaFluor 488</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A-21467</td>
<td>&nbsp;</td>
<tr>
<td>Recombinant human insulin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I2643</td>
<td>&nbsp;</td>
<tr>
<td>Cell lifters</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>29942-118</td>
<td>Cut to fit the wells</td>
</tr>
<tr>
<td>5 ml tubes for FACS</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>60819-138</td>
<td>Fit all BD machines</td>
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quantitative measurement of glut4 translocation to the plasma membrane by flow cytometry

Il glucosio è la principale fonte di energia per il corpo, che richiede una regolazione costante della sua concentrazione nel sangue. Il rilascio di insulina dal pancreas induce assorbimento del glucosio da parte dei tessuti insulino-sensibili, in particolare il cervello, muscolo scheletrico e adipociti. I pazienti affetti da diabete di tipo 2 e / o l&#39;obesità spesso sviluppano resistenza all&#39;insulina e sono in grado di controllare i loro omeostasi del glucosio. Nuove conoscenze sui meccanismi di resistenza all&#39;insulina può fornire nuove strategie di trattamento per diabete di tipo 2. La famiglia di trasportatori del glucosio GLUT è composto da tredici membri distribuiti su diversi tessuti in tutto il corpo 1. Trasportatore del glucosio di tipo 4 (GLUT4) è il principale trasportatore che l&#39;assorbimento di glucosio media da parte dei tessuti insulino-sensibili, come il muscolo scheletrico. Su legame dell&#39;insulina al suo recettore, vescicole contenenti GLUT4 traslocare dal citoplasma alla membrana plasmatica, inducendo l&#39;assorbimento del glucosio. Ridotta traslocazione GLUT4 è una delle cause della resistenza all&#39;insulina nei diabete di tipo 2 2,3. La traslocazione di GLUT4 dal citoplasma alla membrana plasmatica possono essere visualizzati mediante immunocitochimica, utilizzando fluoroforo-coniugati GLUT4 anticorpi specifici. Qui, descriviamo una tecnica per quantificare gli importi totali di GLUT4 traslocazione alla membrana plasmatica delle cellule durante un arco temporale scelto, citometria a flusso. Questo protocollo è rapido (meno di 4 ore, compresi l&#39;incubazione con l&#39;insulina) e consente l&#39;analisi di un minimo di 3.000 cellule o fino a 1 milione di cellule per ogni condizione in un singolo esperimento. Si basa su anti-GLUT4 anticorpi diretti contro un epitopo esterno del trasportatore che si legano ad esso non appena viene esposto al mezzo extracellulare dopo traslocazione alla membrana plasmatica.

Watch this Scientific Journal Video about Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry at JoVE.com

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Questo protocollo descrive una tecnica rapida per quantificare la traslocazione di GLUT4 dal citoplasma alla membrana plasmatica delle cellule mediante citometria di flusso.

Misurazione quantitativa della traslocazione GLUT4 sulla membrana plasmatica mediante citometria di flusso

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

quantitative-measurement-glut4-translocation-to-plasma-membrane-flow

Research

JoVE Journal

Biology

26.3K Views.  Baylor College of Medicine. Questo protocollo descrive una tecnica rapida per quantificare la traslocazione di GLUT4 dal citoplasma alla membrana plasmatica delle cellule mediante citometria di flusso.

Video: Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

Misurazione delle proteine ​​plasmatiche Tariffe endocitico membrana da biotinilazione reversibile

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Ricerca

Biologia

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP-FCM: Immunoprecipitazione rilevato mediante citometria di flusso

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

Purificazione Citometria a flusso di cellule di topo Meiotic

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

Mappatura diffusione molecolare nella membrana plasmatica da parte di più-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Ottimizzato Metodi di modellazione e colorazione proliferazione per il monitoraggio divisione cellulare utilizzando coloranti Tracking Cellulari

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

Analisi in tempo reale di Trasporti retinolo da parte del recettore di membrana di proteine ​​plasmatiche Retinol Binding

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

Immunolocalizzazione della membrana esterna proteine ​​mediante citometria di flusso di mitocondri isolati

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

Endoreduplicazione tramite flusso Cytometry di tubero isolato protoplasti di misura

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.

Misura sensibile di Mitophagy di flusso Cytometry utilizzando il pH-dipendente Reporter fluorescente mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

Misurazione di alto-rendimento di Dictyostelium discoideum Macropinocitosi tramite flusso Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...