1. L&#39;importanza della conta cellulare <ol><li> Conteggio delle cellule è un passo necessario, ma noioso per la coltura cellulare in vitro. </li><li> Le concentrazioni di cellule coerente garantire la riproducibilità sperimentale e precisione. </li><li> Conta delle cellule sono importanti per monitorare la salute delle cellule e tasso di proliferazione, valutando immortalizzazione o trasformazione, per la successiva semina cellule esperimenti, trasfezione o infezione, e prepararsi per saggi cellulari. </li><li> Nonostante la necessità di velocità e la precisione nel conteggio delle cellule, il 71% di 400 ricercatori hanno esaminato le cellule contare utilizzando un emocitometro. Mentre hemocytometry è poco costoso, è laborioso e soggetto a bias di utenti e abuso, che si traduce nella conta inesatti. </li><li> Fonti di errore in hemocytometry includono: distribuzione disomogenea delle cellule nel campione, troppe o troppo poche cellule nel campione, le decisioni soggettive se una data cellula rientra nel settore di conteggio definita, la contaminazione del emocitometro, utente-utente variazione , e la variazione del tasso di riempimento emocitometro. </li><li> Per alleviare la noia associati a conteggio manuale, il 29% dei ricercatori contare le celle con dispositivi automatizzati conteggio delle cellule. Tuttavia, per la maggior parte dei ricercatori, il prezzo associati a questi grandi strumenti da banco è una barriera al loro utilizzo. </li><li> Il contatore di cellule Scettro è un dispositivo automatico portatile che offre l&#39;automazione e la precisione del conteggio Coulter ad un costo relativamente basso. </li><li> Il sistema utilizza il principio Coulter di impedenza a base di rivelazione di particelle in un formato miniaturizzato utilizzando una combinazione di hardware analogico e digitale per il rilevamento, elaborazione del segnale, l&#39;archiviazione dei dati e visualizzazione grafica. </li><li> Il sensore usa e getta è stato progettato con un microfabbricazione, cellula di rilevamento zona che permette la discriminazione in base alle dimensioni delle cellule e il volume delle cellule a livello sub-micron e sub-picolitri risoluzione. </li><li> Avanzata con precisione liquido di gestione dei canali e l&#39;elettronica, il contatore di cellule Scettro visualizza graficamente le statistiche sulla popolazione cella come un istogramma. </li></ol> 2. Preparazione di una singola cellula Sospensione conteggio <ol><li> Per utilizzare il contatore di cellule Scettro, inizia diluendo un&#39;unica sospensione di cellule, in soluzione salina tamponata con fosfato. Una concentrazione di 10,000-500,000 cellule per ml è ottimale. </li><li> Trasferire 100 ml di cellule diluito in una provetta da microcentrifuga 1,5 ml. </li></ol> 3. Esecuzione di conteggio delle cellule usando lo Scettro <ol><li> Accendere il contatore di cellule Scettro tenendo premuto la leva sul retro dello strumento ed attendere la istruzioni sullo schermo ad apparire. </li><li> Quando richiesto, collegare un sensore per la fine dell&#39;unità Scettro con il pannello di elettrodi di rilevamento rivolto verso la parte anteriore dello strumento. </li><li> Sensori scettro composto da una camera di stampaggio di precisione di campionamento, con una zona di rilevamento elettronico e integrato delle cellule-sensing elettrodi. I sensori consentono di discriminare le dimensioni delle cellule a livello sub-micron di risoluzione, e il volume delle cellule a livello sub-picolitri risoluzione. </li><li> Ingegneria sensore coerente, assicura che i volumi di campione pari sono contati e la misurazione delle dimensioni accurate sono fatti. </li><li> Una volta che il sensore è collegato, eseguire ogni passaggio del processo di conteggio in base alla istruzioni sullo schermo come segue: </li><li> Quando lo schermo si legge &quot;Tenere premuto lo stantuffo per iniziare,&quot; spingere il pistone. Lo schermo sarà quindi leggere &quot;immergere il sensore.&quot; Immergere il sensore nella soluzione sospensione cellulare e rilasciare lo stantuffo per aspirare la sospensione cellulare nel sensore. </li><li> Il pistone agisce come un segnale elettrico e prelevare il campione di più volume morto nel sensore. La velocità o la precisione di immergersi non importa come fa con pipettaggio. Questo processo richiederà alcuni secondi. Quando è pronto, lo strumento emette un segnale acustico, e &quot;Sample caricati&quot; sarà visualizzato sullo schermo. </li><li> Una volta che il campione è caricato nel sensore lo ha emesso, attraverso la zona di rilevamento per valutare le dimensioni delle cellule e il volume delle cellule, poi attraverso il microcanali. Il 50 l di campione è contato il sensore dice lo strumento per fermare il conteggio. </li><li> Dopo il conteggio è completa, lo schermo dello strumento leggerà &quot;Count completa. Rimuovere sensore e scartare&quot;. Seguire le operazioni, e rimuovere ed eliminare il sensore. </li><li> Lo strumento quindi visualizzare un istogramma della dimensione della cella o diametro sullo schermo così come la concentrazione di cellule per ml. </li></ol> 4. Analisi dei dati <ol><li> Dopo il conteggio è completa e l&#39;istogramma viene visualizzato sullo strumento, si applicano cancelli premendo il pulsante di commutazione. Selezionare &quot;gating auto&quot; o &quot;ultimo uso&quot; per impostare in modo automatico la porta in base al profilo istogramma, o di utilizzare le porte dal conteggio precedente. </li><li> Indipendentemente dalla scelta, le porte possono essere spostati manualmente per mettere a punto i dati. Premere il pulsante di passare nuovamente per selezionare il cancello di sinistra e di far scorrere laggle per spostare il cancello come desiderato. </li><li> Quindi premere il passare nuovamente per attivare la porta a destra e scorrere la leva per posizionare il cancello giusto. Clicca di nuovo, una nuova concentrazione delle cellule, volume medio delle cellule e diametro sarà visualizzato. </li><li> La ginocchiera può anche essere spostata su entrambi i lati per passare il display tra data, ora, dimensione media delle cellule, ed il volume medio delle cellule. </li><li> Fino a 72 istogrammi possono essere memorizzati sullo strumento stesso. Per caricare i dati ad un personal computer, collegare lo strumento tramite la porta cavo USB sul display. </li><li> Sul PC, fare doppio clic sul software applicativo Scettro per lanciare il software, quindi fare clic su &quot;connect&quot;. Tutti i conteggi si porta automaticamente al software. </li><li> Il Software applicativo Scettro visualizza l&#39;istogramma che ripartisce popolazione della cultura in volume o diametro. I file scaricati possono essere esportati in Microsoft Excel, dove i dati grezzi vengono visualizzati in contenitori di diametro cella specificata o volumi cellulari. </li></ol> 5. Rappresentante Risultati <ol><li> Diciannove diverse linee cellulari sono stati preparati per il conteggio e teorici delle concentrazioni di partenza sono stati determinati utilizzando un contatore Coulter. Queste sospensioni cellulari sono state poi ulteriormente diluito per ottenere sospensioni cellulari a concentrazioni cellulari teorica. </li><li> Il contatore di cellule Scettro è stato poi utilizzato per determinare le concentrazioni cellulari. La figura 1 mostra 5 diluizioni che sono stati misurati per 5 linee di cellule rappresentante insieme alla concentrazione cellulare teorica. </li><li> L&#39;elevato grado di linearità come indicato dai valori di R2, dimostra che il conteggio Scettro è un metodo affidabile per le linee cellulari testate, attraverso una vasta gamma di funzionamento lineare. </li><li> L&#39;accuratezza complessiva relativa di conteggio Scettro è dimostrato nella figura 2, in cui vengono confrontati i dati ottenuti dal conteggio Scettro con conteggi ottenuti da Counting Coulter. </li><li> Il registro di conta delle cellule Scettro è in funzione del log dei conteggi Coulter, e dimostra che, in tutte le linee cellulari testate, conta Scettro corrispondere conta Coulter. </li><li> Conta di 19 linee cellulari sono state effettuate utilizzando un Z2 Coulter Counter, il contatore di cellule Scettro, un contatore automatico visione basata, come Vi-Cell o sistema Innumerevoli, e un hemocytemeter. </li><li> Conteggi sono stati eseguiti secondo le istruzioni del produttore, utilizzando la stessa cellula di partenza sospensione e diluizioni identici. I risultati mostrati in Figura 3 dimostrano che le concentrazioni misurate dal cellulare Scettro contando strettamente corrispondere concentrazioni di cellule teorica con elevata linearità. </li><li> Il coefficiente di variazione percentuale, o CV, è stato calcolato per ogni diluizione di ciascuna linea cellulare per ogni sistema di conteggio. La Figura 4 mostra il CV% medio di oltre 19 linee di cellule rispetto alla concentrazione cellulare e metodo di conteggio. </li><li> All&#39;interno della gamma di funzionamento, il contatore di cellule Scettro è più preciso conteggio emocitometro visione basata, mostrando piccole deviazioni standard e piccoli coefficienti medi di variazione. </li><li> Conteggio scettro è dieci volte più veloce hemocytometry e più velocemente di altri sportelli automatici troppo. Nella Figura 5, SF9, MCF7, e cellule HEK293 sono state contate utilizzando i metodi descritti in questo video. </li><li> Il tempo medio necessario per misurare la concentrazione cellulare è stato registrato. Si noti che hemocytometry è stata eseguita una sola volta per ogni campione. </li><li> Scettro effettua misurazioni in base alle dimensioni della cella. Così qualsiasi eventuale cambiamento morfologico cellulare possono essere catturati nell&#39;istogramma. Culture sano visualizzare un bel distribuzione gaussiana a forma di campana, mentre le cellule malsane, lisare, e provocare un cambiamento nel istogramma a sinistra (Figura 6, pannello a sinistra). </li><li> Allo stesso modo, quando una popolazione pari al 50% cellule morte sono state mescolate con 50% di cellule vive, si sposta istogramma sono stati anche osservati (Figura 6, pannello di destra). </li></ol><img src="/files/ftp_upload/2204/2204fig1.jpg" alt="Figura 1" /><img src="/files/ftp_upload/2204/2204fig2.jpg" alt="Figura 2" /><img src="/files/ftp_upload/2204/2204fig3.jpg" alt="Figura 3" /><img src="/files/ftp_upload/2204/2204fig4.jpg" alt="Figura 4" /><img src="/files/ftp_upload/2204/2204fig5.jpg" alt="Figura 5" /><img src="/files/ftp_upload/2204/2204fig6.jpg" alt="Figura 6" /><img src="/files/ftp_upload/2204/2204fig7.jpg" alt="Figura 7" />

<ol>
	<li>Barghshoon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Counting Survey. , (2009).</li><li>Tucker, K. G., Chalder, S., al-Rubeai, M., Thomas, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+hybridoma+cell+number,+viability,+and+morphology+using+fully+automated+image+analysis.">Measurement of hybridoma cell number, viability, and morphology using fully automated image analysis.</a> Enzyme Microb Technol. 16 (1), 29-35 (1994).</li><li>Houwen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fifty years of hematology innovation: the Coulter principle. , (2003).</li></ol>

Kathleen Ongena, Chandreyee Das, Janet L. Smith, Sonia Gil, e Grace Johnston sono dipendenti di Millipore, che produce gli strumenti utilizzati in questo articolo.

Confrontando le prestazioni del contatore di cellule Scettro ai risultati di altri metodi di conteggio, possiamo concludere che questo nuovo portatile, contaglobuli automatizzato offre conta delle cellule preciso, veloce e affidabile in un ampio campo di funzionamento. La funzionalità di conteggio Scettro è il risultato della tecnologia di precisione costruita incorporato nella punta del sensore e la strumentazione sofisticata conteggio basato sul principio Coulter. La qualità delle prestazioni suggerisce Scettro contare come metodo per aumentare la velocità e migliorare la riproducibilità dei saggi cellulari. Il formato palmare permette noia veloce destra libera conteggio al cofano colture cellulari.

<table border="1"><tbody><tr><td> Nome del materiale </td><td> Azienda </td><td> Numero di catalogo </td><td> Commento </td></tr><tr><td> Scettro 2,0 contatore portatile cella automatizzata con sensori micron 40 Scettro </td><td> EMD Millipore </td><td> PHCC20040 </td><td></td></tr><tr><td> Scettro 2,0 contatore portatile cella automatica con 60 sensori Scettro micron </td><td> EMD Millipore </td><td> PHCC20060 </td><td></td></tr><tr><td> Sensori scettro, 60 micron </td><td> EMD Millipore </td><td> PHCC60050 </td><td> 50 Pack di sensori </td></tr><tr><td> Sensori scettro, 40 micron </td><td> EMD Millipore </td><td> PHCC40050 </td><td> 50 Pack di sensori </td></tr><tr><td> Universal Power Adapter </td><td> EMD Millipore </td><td> PHCCPOWER </td><td> </td></tr><tr><td> Scettro O-Ring Kit </td><td> EMD Millipore </td><td> PHCCOCLIP </td><td> 2 O-ring e un coperchio del filtro </td></tr></tbody></table>

determining cell number during cell culture using the scepter cell counter

Conteggio delle cellule è spesso un passo necessario, ma noioso per la coltura cellulare in vitro. Le concentrazioni di cellule coerente garantire la riproducibilità sperimentale e precisione. Conta delle cellule sono importanti per monitorare la salute delle cellule e tasso di proliferazione, valutando immortalizzazione o trasformazione, per la successiva semina cellule esperimenti, trasfezione o infezione, e prepararsi per saggi cellulari. E &#39;importante che conta delle cellule essere precisi, coerenti e rapidi, in particolare per le misure quantitative di risposte cellulari. Nonostante questa esigenza di velocità e la precisione nel conteggio delle cellule, il 71% di 400 ricercatori hanno esaminato 1 che contare le celle con un emocitometro. Mentre hemocytometry è poco costoso, è laborioso e soggetto a bias di utenti e abuso, che si traduce nella conta inesatti. Hemocytometers sono realizzati in speciale vetro ottico su cui sono caricati sospensioni cellulari in volumi specificati e contati al microscopio. Fonti di errori in hemocytometry includono: distribuzione disomogenea delle cellule nel campione, troppe o troppo poche cellule nel campione, le decisioni soggettive sul fatto che una data cellula rientra nel settore di conteggio definita, la contaminazione del emocitometro, utente-utente variazione , e la variazione del tasso di riempimento emocitometro 2. Per alleviare la noia associati a conteggio manuale, il 29% dei ricercatori contare le celle con il conteggio delle cellule automatizzate dispositivi, questi comprendono visione basati su contatori, sistemi che rilevano le cellule utilizzando il principio Coulter, o citometria a flusso 1. Per la maggior parte dei ricercatori, il principale ostacolo all&#39;utilizzo di un sistema automatizzato è il prezzo associato a queste grandi strumenti da banco 1. Il contatore di cellule Scettro è un dispositivo automatico portatile che offre l&#39;automazione e la precisione del conteggio Coulter ad un costo relativamente basso. Il sistema utilizza il principio Coulter di impedenza a base di rivelazione di particelle 3 in un formato miniaturizzato utilizzando una combinazione di hardware analogico e digitale per il rilevamento, elaborazione del segnale, la memorizzazione dei dati e visualizzazione grafica. La punta di usa e getta è stato progettato con un microfabbricazione, cellula di rilevamento zona che permette la discriminazione in base alle dimensioni delle cellule e il volume delle cellule a livello sub-micron e sub-picolitri risoluzione. Avanzata con precisione liquido di gestione dei canali e l&#39;elettronica, il contatore di cellule Scettro report statistici popolazione di cellule visualizzati graficamente come un istogramma.

Watch this Scientific Journal Video about Determining Cell Number During Cell Culture using the Scepter Cell Counter at JoVE.com

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Il contatore di cellule Scettro è un dispositivo portatile automatizzato che può essere usato per contare le celle, monitorare diametro delle cellule e il volume, e possono essere usate per controllare la salute e la qualità di popolazioni cellulari da una cultura all&#39;altra.

Determinazione del numero delle cellule durante coltura cellulare utilizzando il contatore di cellule Scettro

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

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Biology

37.8K Views.  Millipore Inc. Il contatore di cellule Scettro è un dispositivo portatile automatizzato che può essere usato per contare le celle, monitorare diametro delle cellule e il volume, e possono essere usate per controllare la salute e la qualità di popolazioni cellulari da una cultura all'altra.

Video: Determining Cell Number During Cell Culture using the Scepter Cell Counter

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

Le cellule umane ES: Cultura partire da cellule congelate

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

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Biologia

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Monitoraggio Cambiamenti dei livelli di calcio mitocondriale durante l&#39;apoptosi mediante un sensore di calcio geneticamente codificato

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentivirali-mediata Knockdown Durante Ex Vivo</emEritropoiesi> di cellule staminali emopoietiche

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques6.
Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.
The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.
Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.

The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

Determinazione del conteggio delle cellule dei mammiferi, le dimensioni delle cellule e la salute delle cellule utilizzando il Moxi Z Mini contaglobuli automatizzato

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Cella di rilevamento utilizzando proteine ​​Photoconvertible durante lo sviluppo Zebrafish

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Espianti blastomero al test per l&#39;Impegno cellulare Fate durante lo sviluppo embrionale

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Vivere Imaging di apoptotica delle cellule libera nel corso<em&gt; Drosophila</em&gt; Embriogenesi

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

Confronto di tre diversi metodi per la determinazione proliferazione cellulare in Breast Cancer linee cellulari

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

Vivere la microscopia di fluorescenza delle cellule per osservare i processi essenziali durante la crescita delle cellule microbiche

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

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.