Questo lavoro è stato sostenuto da NIH R01 GM50322 a PM Kane. Gli autori ringraziano il Dr. Rajini Rao, Johns Hopkins University per la fornitura di le di lievito pHluorin plasmidi e di per un consiglio su misure di pH raziometriche, e di il Dr. Gloria A. Martinez Munoz per lavorare fuori questi protocolli per il nostro laboratorio.

1. Misurazione del pH vacuolare In Vivo Uso di BCECF-AM <ol><li> Grow un 50 ml coltura liquida del il ceppo di lievito per essere misurato in il supporto desiderato durante la notte. L&#39;obiettivo è di avere le cellule in di fase mid-log (OD600 (densità ottica a 600 nm) la misurazione di approssimativamente 0,8 per la sospensione). </li><li> A pellet le cellule di lievito per centrifugazione. Risospendere il pellet in 0,6 ml del terreno crescita e trasferimento ad un provetta microcentrifuga che è stato pesato precedenza. A pellet le cellule di nuovo in un microcentrifuga a 2.000 xg per 60 sec. Rimuovere il surnatante il più completamente possibile e poi pesare il pellet di cellule. Risospendere il pellet ad una densità finale di 0,5 g / ml (w / v); una cultura di questo volume vi darà un pellet di cellule di approssimativamente 200 mg, e la 200 microlitri di buffer di sarebbe aggiunto alla dare un volume finale di 400 microlitri. </li><li> Aggiungere BCECF-AM per la sospensione cellulare ad una concentrazione finale di 50 mM da un 12 mM magazzino preparata in DMSO. Mescolare bene e poi incUbate le cellule a 30 ° C per 30 min. su una piattaforma dondolo o di tamburo rullo. </li><li> Mentre le cellule sono incubando, preparare tamponi di calibrazione. Il buffer calibrazione contiene 50 mM MES (2 - (N-morfolino) acid etansolfonico), 50 mM HEPES (4 - (2-idrossietil)-1-piperazineethanesulfonic acid), 50 mM KCl, 50 mM NaCl, 0,2 M acetato ammonio, 10 sodio azide mM, e di 10 mM 2-desossiglucosio, e corretta al fine il pH valori appropriati per il la vostra gamma di calibrazione con NaOH o HCl. (Attenzione: Sodio azide è altamente tossico e deve essere maneggiato con cura.) Per la misura di del pH in cellule di wild-type, ci saremmo preparare parecchi miscele di taratura a pH 5,5 a 6.5, ma per i mutanti attesi di avere una vacuolare pH più alcalino ( vma mutanti), ci saremmo preparare aggiuntivi, tamponi pH più elevati. </li><li> Aliquotare 2 ml di tampone di di calibrazione per ogni del pH in 15 ml provette coniche, e di aggiungere monensin (15 mM magazzino) e di nigericina (2 mM magazzino) per dare concentrazioni finali di 110 mcM monensin e 15 mM Nigericina, respectively. Mescolare bene su un miscelatore vortex. (Attenzione: Sia la monensin e la nigericina sono tossici e di dovrebbero essere maneggiati con cura.) </li><li> Quando il tempo di incubazione BCECF-AM è completato, pellet la sospensione cellulare mediante centrifugazione per 30 sec presso 2.000 xg in una microcentrifuga. Risospendere le cellule è di 1 ml di mezzo di crescita carente glucosio e centrifugare come sopra. Ripetere questa fase di lavaggio una volta, e quindi risospendere il pellet in 200 pl di mezzo di crescita senza di glucosio. Posto sul ghiaccio. </li><li> Aggiungere 20 microlitri della sospensione cellulare a ciascuna provetta calibrazione pH preparato sopra. Incubare a 30 ° C per 30-60 min. su un tamburo rullo. </li><li> Impostare il fluorimetro per misurare alternativamente presso di eccitazione lunghezze d&#39;onda di 450 nm e 490 nM, entrambi i con una lunghezza d&#39;onda di 535 nm emissione di. Impostare la temperatura camera del campione a 30 ° C. Eseguire tutte le misurazioni con agitazione continua della miscela, in il cuvetta. </li><li> Aggiungere 1.96 ml su 1 mM MES (regolata a pH 5 o 7 seconda del pH delle crescita di medio delle cellule &#39;unand il disegno sperimentale). Aggiungere 20 l di sospensione cellulare nella cuvetta. Initiate misure di fluorescenza. Noi raccogliamo entrambi cinetica continuo e di dati tempo di punti d&#39;ancoraggio in differenti esperimenti di. Per dati cinetici continui, prendiamo misurazioni ogni 6 sec per 5 min, quindi aggiungere glucosio per una concentrazione finale di glucosio di 50 mM, e continuare misurazione per 5-10 min. Per le misurazioni singoli di tempo-point, noi generalmente prendiamo misurazioni di presso 1 e di 5 min. dopo aggiunta delle cellule ai della cuvetta, aggiungere glucosio come nelle le misure cinetiche, e poi prendere un&#39;altra misurazione 5 min. dopo addizione glucosio. Queste aggiunte possono essere variate, e componenti addizionali (inibitori, etc) possono anche essere aggiunte. </li><li> Dopo le misure sperimentali sono completi, togliere le provette di calibrazione da 30 ° C incubazione e trasferire l&#39;intero volume 2 ml per ciascun alla cuvetta fluorimetro. Misurare fluorescenza presso le stesse impostazioni (cfr. 1.8) ogni 5 s sopra un totale di 30 sec per ogni campione. </li><li>Esportare dati di fluorescenza a Microsoft Excel. (Per il nostro fluorimetro, questo richiede l&#39;esportazione dei dati come testo in forma delimitato da tabulazioni e l&#39;importazione in Excel.) Procurarsi una curva di calibrazione da parte il calcolo del rapporto di di fluorescenza a 490 nm a 450 nm per ciascun miscela di calibrazione. Il rapporto di fluorescenza viene poi tracciata vs pH per ottenere una curva di calibrazione. </li><li> Convertire i dati sperimentali ai rapporto di fluorescenza e calcolare pH usando la curva standard. PH vacuolar può allora tramato contro tempo (cinetica di modifiche del pH) né confrontato sotto varie condizioni (figura 1) o in differenti ceppi mutanti. </li></ol> 2. Misurazione del pH citosolico In Vivo Uso di Lievito pHluorin <ol><li> Trasforma il ceppo di lievito desiderato con il pHluorin lievito plasmide da parte protocolli standard, e selezionare per la trasformanti su completati minima quantità di terreno uracile carente (SC-uracile). </li><li> Grow un 50 ml coltura liquida delle cellule trasformate in SC-uracile a di fase mid-log (OD <sub&gt; 600 = 0.8 o inferiore). </li><li> Preparare gli standard di calibrazione come descritto per il pH vacuolare, ma buffer di ad una gamma di pH appropriato per misure di pH citosolici, generalmente di pH 6-8. Aliquotare 2 ml di tampone di calibrazione regolata alla pH desiderato in parecchie provette coniche 15 ml. Aggiungi monensin e di nigericina come descritto sopra (1.4) e mescolare bene. </li><li> Raccogliere le cellule per centrifugazione come descritto sopra, e risospendere il pellet in 600 microlitri della mezzo di crescita. Trasferire in un provetta da microcentrifuga pesato, e le cellule pellet per centrifugazione a 5.000 rpm per 30 sec. Risospendere il pellet in 1 ml di mezzo di crescita senza il glucosio (SC-uracile,-glucosio), le cellule a pellet di nuovo e ripetere. Dopo che il centrifugazione finale, rimuovere il surnatante come a fondo il più possibile e pesare il pellet di cellule. Risospendere celle a una densità finale di 0,5 g / ml in SC-uracile con nessuna glucosio. </li><li> Aggiungere 20 microlitri di sospensione cellulare a ciascuna provetta di tampone di calibrazione e di mescolare bene su un miscelatore vortex. Incubare a 30 ° C su un rotore tamburo di filatura per 60 min. </li><li> Impostare up il fluorimetro per la eccitazione a lunghezze d&#39;onda 405 e 485 nm e di un lunghezza d&#39;onda di 508 nm emissione di. Procedere con punto di singola volta o di misurazioni di cinetiche e la aggiunta di glucosio come descritto sopra per la misurazione BCECF. </li><li> Misurare fluorescenza dei campioni di taratura, e costruire una curva di calibrazione da per BCECF (cfr. 1,10-1,11). Un appezzamento di rapporto di di fluorescenza vs del pH è lineare sopra la gamma di la maggior parte delle misure di pH citosoliche (pH 6.0-8.0) 9, in modo sperimentali le misure del rapporto di fluorescenza sono facilmente convertiti a pH. </li></ol>

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Noi abbiamo utilizzato questi protocolli per affrontare un certo numero di gli aspetti della pH omeostasi. Ad esempio, noi abbiamo confrontato le risposte citosoliche e di pH di cellule mutanti wild-type e V-ATPasi-carente 4,5. Abbiamo inoltre esaminato gli effetti delle condizioni di crescita alterati, di pH particolarmente extracellulare, sulla risposta del pH vacuolare al glucosio 3. È importante sottolineare che, le risposte che osserviamo sono entrambi coerenti con altri metodi di misurazione...

pH omeostasi è un processo dinamico e di altamente regolato in tutti gli organismi 1,2. Processi biochimici sono strettamente regolati da pH, e di ambienti di intracellulari sono sintonizzati ai range di di pH strette per consentire attività ottimale delle le enzimi residenti. Tuttavia, intracellulare pH omeostasi può essere contestata da rapidi cambiamenti in pH ambientale, turni di metabolici, e di alcuni vie di segnalazione. In aggiunta, pH intracellulare può servire di per sé come un segnale importante. Infine, molti organelli mantengono valori pH lumenal che sono distinti dal citosol circostante e essenziale per le funzioni organello-specifici. Le lieviti Saccharomyces condivide cerevisiae un certo numero di meccanismi omeostasi pH con con eucarioti superiori 2. In gli organelli acide del il pathway endocitica / lisosomiale, pH è controllato in primo luogo dalla altamente conservata vacuolare protone-translocating ATPasi (V-ATPasi), agendo in tandem con molti scamgers dipendenti dal gradiente di di pH. Tutte le cellule eucariotiche hanno anche meccanismi di esportazione protonica. In funghi e piante, un secondo, pompa di protonica distinto a livello della membrana plasmatica, Pma1, le esportazioni protoni metaboliche e si crede di essere il maggiore fattore determinante di pH citosolica e di potenziale di membrana plasmatica. La flessibilità genetica di S. cerevisiae e la sua importanza commerciale, hanno reso esso un modello molto interessante e importante per lo studio pH l&#39;omeostasi 2. In Oltre ad essere i driver di primari di organello acidificazione, V-ATPasi di sono altamente regolamentati enzimi di e la il nostro laboratorio di è interessato a meccanismi di regolazione V-ATPasi la comprensione. Verso questo obiettivo, abbiamo sono state utilizzando in misure di pH in vivo di pH vacuolare e di citosolica: 1) a monitorare le risposte al mutevoli condizioni extracellulari, come ad esempio deprivazione glucosio e readdition, 2) ai esaminare gli effetti di mutazioni che compromesso di attività V-ATPasi, e di 3) per esplorare la coordinamento di degli organelli e di al plasma a membrana pompe protoniche 3-5. Questi esperimenti divennero possibile solo attraverso lo sviluppo di solidi indicatori di pH raziometrici suscettibili di utilizzare in cellule di lievito. . Pianta et al primo luogo ha mostrato che BCECF (2&#39;7&#39;-Bis-(2-carbossietil) -5 - (e 6)-carbossifluoresceina), che è stato usato ampiamente per misurare il pH citosolico in cellule di di mammifero, accumula nel vacuolo lievito anziché il citosol 6. Questa differenza nella BCECF localizzazione è stata attribuita ad le molte enzimi idrolitici in il vacuolo, che sono probabilmente responsabile per il clivaggio dell&#39;estere metilico acetossi da BCECF-AM (estere acetossimetil di BCECF) e ritenzione vacuolare 6. Ali et al. 7 ulteriormente sviluppato di misura del pH vacuolare usando BCECF e adattati queste misurazioni per un formato di lettore di piastre di fluorescenza. Brett et al. Introdotta lievito pHluorin come un mezzo di misurare pH citosolico in lievito da esprimendo un r plasmide-borneatiometric GFP pH-sensibile al 8 sotto controllo di un lievito-specifico promotore 9. La spettri di eccitazione di entrambi BCECF e di lievito pHluorin sono sensibili ai pH, in modo da essi sono utilizzati come indicatori di pH raziometrici in cui il rapporto di di fluorescenza presso due lunghezze d&#39;onda di eccitazione, misurati ad una singola lunghezza d&#39;onda di emissioni, fornisce una misura di pH 8,10. Questi sensori di pH vacuolari e di citosolica di lievito sono stati utilizzati per entrambe le misurazioni a cella singola e di basato sulla popolazione. Misurazioni monocellulari 6,11 sono eseguite da microscopia a fluorescenza e analisi di immagine. Fluorescenza vacuolar o citosolico presso i due lunghezze d&#39;onda è misurato per ogni cella. Le misurazioni basati sulla popolazione vengono eseguite sia in un lettore di micropiastra con adeguate capacità di fluorescenza o in un fluorimetro. Noi abbiamo generalmente fatto i nostri misurazioni di in un fluorimetro, perché fornisce un facile accesso per aggiunta di componenti di come ad esempio glucosio durante condizionatamisurazioni di cinetiche continuo. I nostri protocolli di di laboratorio attuali per la misurazione di del pH vacuolare e di citosolica sono elencati di qui di seguito; entrambi sono anche facilmente adattati a saggi di per micropiastre.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Spectrofluorometer</td>
 <td>Horiba Jobin Yvon</td>
 <td>Model Fluoromax-4</td>
 <td>Temperature control and stirring capability are desirable.</td>
 </tr>
 <tr>
 <td>BCECF-AM</td>
 <td>Invitrogen/Molecular Probes</td>
 <td>B1150</td>
 <td>Prepare a 12 mM stock in dry DMSO, store as aliquots at -20 &deg;C </td>
 </tr>
 <tr>
 <td>monensin</td>
 <td>Sigma</td>
 <td>M5273</td>
 <td>Toxic. </td>
 </tr>
 <tr>
 <td>nigericin</td>
 <td>Sigma</td>
 <td>N7143</td>
 <td>Toxic. </td>
 </tr>
 <tr>
 <td>MES</td>
 <td>Sigma</td>
 <td>M8250</td>
 <td></td>
 </tr>
 </tbody>
</table>

measurement of vacuolar and cytosolic ph in vivo in yeast cell suspensions

Vacuolare e di pH citosolica sono altamente regolamentati in cellule di lievito e di occupano un ruolo centrale nella complessiva omeostasi pH. Noi descriviamo i protocolli per la la misurazione raziometrica di del pH in vivo usando di pH-sensibili fluorofori localizzate al il vacuolo o di citosol. PH vacuolar viene misurata usando BCECF, che localizza al la vacuolo in lievito quando introdotto in cellule nella sua forma estere acetossimetil. PH citosolico è misurata con un GFP pH-sensibile al espressa sotto controllo di un promotore lievito, lievito pHluorin. Metodi di misura della rapporti di fluorescenza in sospensioni cellulari lievito negli una fluorimetro sono descritte. Attraverso questi protocolli, le misurazioni singole punto di tempo di pH in condizioni diversi o in differenti mutanti di lievito sono stati confrontati e di le variazioni di pH nel corso del tempo sono stati monitorati. Questi metodi sono stati anche adattati a una formato di lettore di piastre di fluorescenza per gli esperimenti di ad alta-di throughput. Vantaggi di misure di pH raziometrici oltre altri apsono anche descritti procci attualmente in uso, i potenziali problemi sperimentali e soluzioni, e le prospettive per uso futuro di queste tecniche.

La Figura 1 presenta i dati di pH vacuolari ottenuti su cellule di lievito wild-type coltivate in ricco di medie (di lievito estratto di-peptone-destrano; YEPD) memorizzati nel buffer pH 5 con 50 mM MES. Abbiamo Spesso cresciamo le cellule in mezzo tamponato perché il pH del mezzo può cambiare abbastanza drammaticamente durante la crescita durante la notte, particolarmente per terreno minimo, e abbiamo trovato che il pH del mezzo di crescita può influenzare risposte di pH vacuolari 3. Tutt...

Watch this Scientific Journal Video about Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions at JoVE.com

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

PH vacuolare e di citosolica può essere misurata in lieviti vivi (<em&gt; S. cerevisiae</em&gt;) Cellule utilizzando tinture fluorescenti raziometrici localizzate a specifici compartimenti cellulari. Noi descriviamo procedure per la misurazione del pH vacuolare di con BCECF-AM, la quale localizza al la vacuolo nel lievito, e del pH citosolico con un citosolica raziometrico GFP pH-sensibile al (lievito pHluorin).

Misurazione di pH vacuolare e di Cytosolic<em&gt; In Vivo</em&gt; In Lievito Cell Sospensioni

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

measurement-vacuolar-cytosolic-ph-vivo-yeast-cell

Research

JoVE Journal

Biology

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

19.7K Views.  SUNY Upstate Medical University. PH vacuolare e di citosolica può essere misurata in lieviti vivi ( S. cerevisiae) Cellule utilizzando tinture fluorescenti raziometrici localizzate a specifici compartimenti cellulari. Noi descriviamo procedure per la misurazione del pH vacuolare di con BCECF-AM, la quale localizza al la vacuolo nel lievito, e del pH citosolico con un citosolica raziometrico GFP pH-sensibile al (lievito pHluorin).

Video: Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

In vivo micro-circolazione di misura nel muscolo scheletrico da Intra-vitale Microscopia

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Ricerca

Biologia

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

Misurazione quantitativa della Invadopodia-mediata proteolisi della matrice extracellulare in singoli contesti e pluricellulari

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

La misurazione simultanea di pH in endocitosi e citosoliche scomparti nelle cellule viventi usando Microscopia confocale

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

La misurazione dei livelli Heme sintesi in cellule di mammifero

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Misure calcio citosolico nelle cellule epiteliali renali mediante citometria di flusso

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Analisi delle sospensioni cellulari isolate da tessuti solidi da Spectral citometria a flusso

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Ad alta risoluzione Imaging e analisi dei singoli microtubuli astrali Dynamics in germogliamento lievito

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

Microscopia 4D del lievito

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Effetto di proteine fluorescenti su Fusion partner utilizzando saggi di tossicità di Polyglutamine in lievito

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Misurazione dei tassi di consumo di ossigeno in intatto Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...