Lo studio è supportato da US Public Health Service Concessione DK-081527, 072473 e DK-DK-20595 per l&#39;Università di Chicago Diabetes Centro di Ricerca e Formazione (Animal Models core), e un dono da parte della Fondazione Famiglia Kovler.

Parte 1: Preparazione dei campioni <ol><li> Posizionare il mouse al microscopio dissezione, rimuovere l&#39;intero pancreas con il duodeno e la milza ancora attaccato, e posizionare gli organi su un vetrino pre-pesati. Rimuovere il duodeno, tagliando il suo tessuto connettivo lungo il lato dove è legato al pancreas. Tagliare e rimuovere il grasso in eccesso milza e il pancreas fino a quando il pancreas è completamente isolato nella diapositiva. (Grasso del pancreas è riconoscibile per il colore bianco, come differenziate dal colore leggermente più scuro e conciatore di tessuto pancreatico.) </li><li> Posizionare un coprioggetto (copertura in vetro di grandi dimensioni 50x75mm; standard di copertura in vetro 25x75mm) sul pancreas in modo che sia interamente coperto. Usare un peso (ad esempio un libro pesante) per appiattire il pancreas tra la diapositiva e la sua copertina. A seconda del peso, appiattendo il pancreas dura circa cinque minuti. </li><li> Togliere il peso. Se il pancreas è ben appiattito contro la diapositiva, è posto nel 4% paraformaldeide (PFA) a 4 ° C durante la notte. Al mattino, rimuovere il coprioggetto e ben esporre il pancreas intero PFA durante la giornata (6-8 ore). Se il pancreas non è ben fisso, permettono di rimanere nel PFA più a lungo, forse un giorno all&#39;altro. Trasferire i vetrini al contenitore con l&#39;1% Triton X-100 in tampone fosfato (PBS) durante la notte. Il giorno dopo, trasferimento le diapositive al contenitore di saccarosio saturi per 1-2 giorni. Poi trasferire le diapositive in un contenitore del 100% glicerolo fino a quando il tessuto viene cancellata, che permette la risoluzione ottimale di segnali fluorescenti; compensazione tessuto assume giorni circa 1-3. (I segnali fluorescenti possono persistere per settimane e anche mesi, ma l&#39;imaging deve essere eseguita subito dopo il tessuto viene cancellata per la risoluzione ottimale.) </li><li> Conservare il pancreas in una zona buia e fredda al fine di preservare l&#39;organo ed i suoi segnali fluorescenti. </li></ol> Parte 2: immagini e quantificare le cellule beta nel pancreas zona intatta <ol><li> Uso di pinze o guanti, prendere lo scorrimento fuori dal contenitore con il 100% glicerolo. Preparare il vetrino per l&#39;imaging con l&#39;annientamento fuori glicerolo in eccesso ed esclusivamente la pulizia del retro del vetro di copertura con etanolo. </li><li> Aprire il software StereoInvestigator (MicroBrightField, Williston, VT) e selezionare la lente 2x obiettivo per i topi di imaging pancreas età superiore ai 3 settimane su grandi lastre di vetro, o le lenti dell&#39;obiettivo 10x per i topi di imaging pancreas più giovane di 3 settimane pressato tra due lamelle 25 millimetri. Visualizzare l&#39;immagine impostando a vivere immagine (Immagine → Foto Live). Selezionare un tempo di esposizione che mostra chiaramente la GFP-tagged beta-cellule. (Sottoesposizione dell&#39;immagine esclude piccoli isolotti e beta-cellule, mentre sovraesporre l&#39;immagine crea ulteriori falsi segnali.) </li><li> Clicca un punto qualsiasi dello schermo per stabilire un punto di riferimento e procedere per disegnare un contorno di tutto il pancreas intero. Avviare il processo virtuale Slice (Immagine → Acquisire fetta virtuale). Determinare e selezionare le migliori cioè sul piano focale, l&#39;aereo con il maggior numero di isole visibili e concentrati e beta-cellule della Z scorrendo la manopola di messa a fuoco. Una volta scelto, StereoInvestigator acquisisce automaticamente l&#39;immagine visualizzata sullo schermo. Con la fase motorizzata, la funzione Slice virtuale cattura sequenziale ciascun pannello ottico come immagine distinta all&#39;interno del contorno disegnato (fig. 1A) e quindi unisce tutti come un&#39;unica immagine fusa (Fig. 1B). Di imaging fetta virtuale utilizza una configurazione epifluorescente filtri cioè widefield che permette alla fotocamera di catturare tutti i segnali presenti ad una certa profondità, compresi quelli non perfettamente a fuoco, in modo che la fetta finale virtuale è un sistema integrato di immagine bidimensionale che non è al massimo proiezione dalla ricostruzione tridimensionale del pancreas intero. Media virtuale scansione in tempo Slice è di 30 minuti per ogni campione, ma può richiedere fino a un&#39;ora per un pancreas di grandi dimensioni. </li><li> Dopo aver salvato l&#39;immagine, inizia la sua analisi aprendo l&#39;immagine con ImageJ. Quando finisce di caricare l&#39;immagine e appare sullo schermo, aprire ed eseguire il Virtual macro analisi Slice ( <a href="http://www.jove.com/files/ftp_upload/1970/Supplementary_material 1.pdf">supplementare materiale 1</a> ). </li><li> Seguire passo per passo le istruzioni fornite dal macro. Eliminare gli artefatti indesiderati creato dalla diffusione della luce con la &quot;bacchetta magica&quot; e &quot;Select Area&quot; strumenti. Controllare e sottrarre sfondo inutili dall&#39;immagine. Determinare il raggio del più grande isolotto con lo strumento &quot;Linea&quot; in modo che la caratteristica conseguente Rolling Ball può rimuovere efficacemente la luce autofluorescenza e dispersi. Test e selezionare il più appropriato &quot;Bassa Soglia&quot; per catturare i segnali fluorescenti di isolotti e cluster di beta-cellule (&lt;3x10 4 micron 2). Test e selezionare il più appropriato &quot;Soglia alta&quot; per catturare i segnali fluorescenti di isolotti. Eseguire la quantificazione del Slice virtuali, che misureràe produrre un elenco di zona isolotto, il perimetro, circolarità, e il diametro di Feret. </li></ol><img src="/files/ftp_upload/1970/1970Fig1A.jpg" alt="figura 1a" /> Figura 1A virtuale immagine Fetta di analisi l&#39;intera distribuzione delle beta-cellule in un pancreas adulto intatto Immagini dei singoli pannelli ottico di un pancreas topo maschio a 10-settimana presa con un obiettivo 2x. Le immagini comprendono l&#39;intera distribuzione di isolotti, anche piccoli gruppi di beta-cellule (&lt;10 cellule). <img src="/files/ftp_upload/1970/1970Fig1B.jpg" alt="figura 1b" /> Figura 1B virtuale immagine Fetta di analisi l&#39;intera distribuzione delle beta-cellule in un pancreas adulto intatto Una fetta unificato virtuale con il pancreas dorsale a destra e il pancreas ventrale a sinistra. Barra di scala è 5 mm. Rappresentante Risultati Preparazione di un pancreas avanzato per l&#39;imaging fetta virtuale consentirà di quantificare efficace di tutte GFP-tagged beta-cellule del pancreas in un intero come immagine fetta virtuale con isolotti chiare e distinte. Successo di imaging fetta virtuale del pancreas intero in situ fornisce i mezzi per esaminare e quantificare le beta-cellule, non solo zona isolotto individuale e totale, ma anche distribuzione delle dimensioni delle isole, l&#39;analisi regionale, e modelli di crescita pure. Mentre ImageJ mantiene la capacità di rilevare tutti i beta-cellule contemporaneamente l&#39;analisi delle immagini Slice virtuali, può anche controllare quali beta-cellule per quantificare limitando l&#39;analisi alle dimensioni isolotto certo (per area o diametro), o per posizione. Una analisi standard esclude per esempio gli artefatti, detriti più piccoli di un singolo beta-cellule (&lt;170 micron 2) e registra l&#39;area, il perimetro (la distanza che circonda una zona), la circolarità (un grado di rotondità, dove 1.0 rappresenta un cerchio perfetto), e del diametro Feret (la distanza più lunga in uno spazio) per ogni isolotto rilevato. Analisi fetta virtuale è in grado di migliorare le immagini fetta virtuale utilizzando segmentazione Watershed, che distingue e separa isolotti sovrapposizione in isolotti individuali in base al loro forme. L&#39;analisi produce inoltre una serie di immagini dettagliate; questo include le maschere delle immagini sotto le due intensità bassa e alta (Fig. 1C), uno schema di tutte le isolette presunte, che sono singolarmente numerati ed elencati in una tabella di informazioni corrispondenti (Fig. . 1D), e l&#39;immagine originale fetta virtuale (Fig. 1B). Si noti che polarizzazione potenziale tecnico per la scelta dei valori di soglia possono inclinare la quantificazione delle isole includendo luce circostante gratuita strutture di grandi dimensioni, o riducendo il numero totale di isole, i segnali deboli soprattutto da piccole particelle. <img src="/files/ftp_upload/1970/1970Fig1C.jpg" alt="figura 1c" /> Figura 1C virtuale immagine Fetta di analisi l&#39;intera distribuzione delle beta-cellule in un pancreas adulto intatto Una maschera delle particelle fluorescenti nella fetta virtuale. Codici colori: [1] blu: fluorescenza a bassa intensità; [2] verde: fluorescenza intensità intermedia; e [3] rosso: a fluorescenza ad alta intensità. <img src="/files/ftp_upload/1970/1970Fig1D.jpg" alt="figura 1d" /> Figura 1D virtuale Fetta di analisi delle immagini l&#39;intera distribuzione delle beta-cellule in un pancreas adulto intatto Quantificazione degli isolotti singoli / gruppi di beta-cellule. Si noti che ogni isolotto (tra cui piccoli gruppi di cellule beta) è numerata, con le sue informazioni in un grafico corrispondente. Quattro parametri sono stati presi per ogni struttura: (1) zona; (2) perimetro, (3) circolarità: un grado di rotondità, dove il numero 1.0 rappresenta un cerchio perfetto, e (4) Feret di diametro: la distanza più lunga all&#39;interno di un&#39;area. Si noti che # 706 visualizza la risoluzione di analisi con una superficie di poche cellule beta. Segmentazione spartiacque rileva gruppi di isolotti adiacenti, come quella mostrata, e si distingue opportunamente come isolotti distinti (isolotti 1554, 1555, e 1556). <img src="/files/ftp_upload/1970/1970Fig1E.jpg" alt="figura 1e" /> Figura 1E virtuale immagine Fetta di analisi l&#39;intera distribuzione delle beta-cellule in un pancreas adulto intatto 3-dimensionale trama dell&#39;analisi fetta virtuale, con assi di area, perimetro, e il diametro di Feret. Ogni punto rappresenta un isolotto.

<ol>
	<li>Miller, K., Kim, A., Kilimnik, G., Moka, U., Jo, J., Periwal, V., Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+formation+during+the+neonatal+development+in+mice.">Islet formation during the neonatal development in mice.</a> PloS One. 4, E7739-E7739 (2009).</li><li>Kilimnik, G., Kim, A., Jo, J., Miller, K., Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+pancreatic+islet+distribution+in+situ+in+mice.">Quantification of pancreatic islet distribution in situ in mice.</a> Am J Physiol Endocrinol Metab. 297, E1331-E1338 (2009).</li><li>Hara, M., Wang, X., Kawamura, T., Bindokas, V. P., Dizon, R. F., Alcoser, S. Y., Magnuson, M. A., Bell, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+with+green+fluorescent+protein-labeled+pancreatic+beta+-cells.">Transgenic mice with green fluorescent protein-labeled pancreatic beta -cells.</a> Am J Physiol Endocrinol Metab. 284, E177-E183 (2003).</li><li>Hara, M., Dizon, R. F., Glick, B. S., Lee, C. S., Kaestner, K. H., Piston, D. W., Bindokas, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+pancreatic+beta-cells+in+the+intact+pancreas.">Imaging pancreatic beta-cells in the intact pancreas.</a> Am J Physiol Endocrinol Metab. 290, E1041-E1047 (2006).</li></ol>

Nessun conflitto di interessi dichiarati.

L&#39;analisi delle immagini fetta virtuale di isole e cellule beta nel pancreas cluster intatto offre una grande vista l&#39;intera distribuzione delle isolette. Sviluppi e cambiamenti nella distribuzione delle isole totale nel corso del tempo può quindi essere studiati e confrontati. Un esempio è dimostrato nella nostra analisi della formazione di isole pancreatiche (1). Un analisi completa delle neonatale pancreas topi in vari momenti (P1-P21) ha dimostrato che le isole sono formate da fissione. Mentre il beta-prol...

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in situ quantification of pancreatic beta-cell mass in mice

Tracing cambiamenti di popolazioni di cellule specifiche in materia di salute e malattia è un obiettivo importante della ricerca biomedica. Il processo di monitoraggio pancreatiche beta-proliferazione delle cellule e la crescita delle isole è particolarmente impegnativo. Abbiamo sviluppato un metodo per catturare la distribuzione delle beta-cellule del pancreas intatto di topi transgenici con fluorescenza tag beta-cellule con una macro scritta per ImageJ (rsb.info.nih.gov / ij /). In seguito a dissezione del pancreas e di compensazione dei tessuti, il pancreas intero è catturata come una fetta virtuale, dopo di che la GFP-tagged beta-cellule sono esaminati. L&#39;analisi include la quantificazione del totale delle cellule beta zona, il numero di isole pancreatiche e distribuzione delle dimensioni con riferimento a parametri specifici e le posizioni per ogni isola e per piccoli gruppi di beta-cellule. L&#39;intera distribuzione delle isole possono essere tracciati in tre dimensioni, e le informazioni dalla distribuzione delle dimensioni e della forma di ogni isolotto consente un confronto quantitativo e qualitativo dei cambiamenti nella complessiva delle cellule beta zona a colpo d&#39;occhio.

Watch this Scientific Journal Video about In situ Quantification of Pancreatic Beta-cell Mass in Mice at JoVE.com

In situ Quantification of Pancreatic Beta-cell Mass in Mice

Il protocollo seguente delinea il processo di dissezione del pancreas per l&#39;imaging fetta virtuale, e la successiva quantificazione di tutte le GFP-tagged beta-cellule del pancreas intero.

Quantificazione in situ di cellule beta pancreatiche Messa in topi

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic ...

in-situ-quantification-of-pancreatic-beta-cell-mass-in-mice

Research

JoVE Journal

Biology

In situ Quantification of Pancreatic Beta-cell Mass in Mice

12.1K Views.  University of Chicago. Il protocollo seguente delinea il processo di dissezione del pancreas per l'imaging fetta virtuale, e la successiva quantificazione di tutte le GFP-tagged beta-cellule del pancreas intero.

Video: In situ Quantification of Pancreatic Beta-cell Mass in Mice

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

Il trapianto di isole pancreatiche nella capsula renale di topi diabetici

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

Ricerca

Biologia

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Quantificazione dei γH2AX Foci in risposta alle radiazioni ionizzanti

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

Assistita da computer su larga scala di visualizzazione e quantificazione della Messa di isole pancreatiche, distribuzione delle dimensioni e architettura

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

Quantificazione delle proteine ​​Utilizzando Arricchimento Peptide immunoaffinità Accoppiato con spettrometria di massa

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

Valutazione della funzione muscolare del muscolo estensore lungo delle dita<em&gt; Ex vivo</em&gt; E Muscolo tibiale anteriore<em&gt; In situ</em&gt; In Mouse

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualizzazione e l&#39;analisi di molecole di mRNA mediante fluorescenza<em&gt; In Situ</em&gt; Ibridazione in<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Reazione selezionato Monitoraggio spettrometria di massa per Absolute Protein Quantificazione

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Risoluzione Quantificazione alta del cristallino cellulosa accumulo in<em&gt; Arabidopsis</em&gt; Roots a Monitor tessuto-specifica delle cellule parete Modifiche

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

Osservazione e quantificazione dei telomeri e sequenze ripetitive Usando fluorescenza<em&gt; In Situ</em&gt; Ibridazione (FISH) con sonde PNA in<em&gt; Caenorhabditis elegans</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.