Il lavoro descritto in questo manoscritto è stato reso possibile da un finanziamento della Fondazione Patrick F. Taylor.

1. Cromosoma raccolta di cellule aderenti <ol><li> Protocollo standard <ol><li> Crescere le cellule in base alle specifiche condizioni di coltura delle cellule. Quando le cellule hanno raggiunto la fase logaritmica (80% di confluenza), aggiungere 10 microlitri / ml di Colcemid al pallone di coltura cellulare. Si raccomanda un minimo di 2 x 10 6 cellule. </li><li> Incubare le cellule a 37 ° C in un incubatore CO 2 5% per 45 min. Usando una pipetta sterile, trasferire file multimediali da cellule in una provetta da 15 ml. Mettere da parte. </li><li> Lavare delicatamente le cellule aggiungendo 2 ml di HBSS tampone nel pallone. Tampone Swirl e poi rimuovere con una pipetta. Scartare. </li><li> Aggiungere 1 ml di tripsina, assicurando che copre l&#39;intera superficie del pallone. Lasciare solo le cellule in tripsina per circa 2 min. Una volta che la maggior parte delle cellule sono distaccato, pipettare i media nel tubo conico indietro sulle cellule. </li><li> Trasferire la sospensione cellulare in aliquote da 10 ml in provette da 15 ml coniche.Centrifugare a 200 xg per 10 min. Rimuovere il surnatante e risospendere il pellet. </li><li> Aggiungere 10 ml di 0,075 M KCl preriscaldata a 37 ° C al pellet rimasto nel tubo conico. Tubo Vortex a velocità media per mescolare KCl e le cellule. </li><li> Incubare le cellule a 37 ° C per 10 min. Centrifugare a 200 xg per 5 min a 25 ° C. Rimuovere il surnatante (fino a circa 0,5 ml resti) e risospendere pellet. </li><li> Aggiungere con cautela 5 ml di fissativo fresco di Carnoy (3:1 rapporto di metanolo: acido acetico glaciale) alle cellule, mentre vortex. Aggiungere quindi 5 ml più di fissativo, senza vortex per un totale di 10 ml. </li><li> Centrifugare a 200 xg per 5 min. Rimuovere le cellule surnatante e sospendere nuovamente. Aggiungere 5 ml di fissativo ad ogni provetta. </li><li> Centrifugare a 200 xg per 5 min. Rimuovere le cellule surnatante e sospendere nuovamente. Aggiungere 5 ml di fissativo ad ogni provetta. Le cellule possono essere conservate a 4 ° C fino ad un anno. </li></ol></li><li> Modifica del protocollo: harvesti cromosoming di linee cellulari linfoblastoidi <ol><li> Crescere le cellule in base alle specifiche condizioni di coltura delle cellule. (Usa pallone appropriato per la quantità di cellule in coltura). Quando le cellule hanno raggiunto la fase logaritmica (circa 2 giorni dopo le cellule sono split), aggiungere 10 microlitri / ml di Colcemid al pallone di coltura cellulare. </li></ol></li><li> Modifica del protocollo: Cromosoma raccolta da sangue intero Fitoemoagglutinina (PHA), una lectina derivato dal fagiolo rosso, è un potente mitogeno per le cellule T umane 16. 72 ore dopo l&#39;aggiunta di PHA alla cultura, circa il 45% delle cellule sono in fase S. Questo rappresenta il picco di attività mitotica, ed è il punto ottimale in cui per raccogliere per gli studi cromosomici. Altri mitogeni come pokeweed (1-10 mg / ml) possono essere utilizzati nell&#39;analisi cellule B 17,18. <ol><li> Raccogliere almeno 1 ml di sangue intero in una parte superiore del tubo di sodio eparina verde. Usare entro 3 giorni dopo la raccolta. Sangue Conservare a temperatura ambiente delle Nazioni Unitetil pronto all&#39;uso. </li><li> Aliquotare 0,25 ml di sangue intero in 10 ml di mezzi RPMI completo contenente L-glutammina (siero fetale bovino 20%, 1% di penicillina / streptomicina, 1% fungizone, e 1% PHA). Coltura a 37 ° C con 5% di CO 2. </li></ol></li><li> Modifica del protocollo: Cromosoma raccolta dal midollo osseo Analisi citogenetica sul midollo osseo è utile in molti disturbi ematologici maligni come l&#39;osservazione di un clone cromosomicamente anormale può essere diagnostica per un tipo specifico di leucemia. Studi cromosomiche sono utilizzati anche per valutare la progressione della malattia, come l&#39;insorgenza di crisi blastica e risposta al trattamento. Poiché le cellule del midollo osseo sono attivamente dividono, nessuno stimolo mitogeno è necessaria 9,19. Per le leucemie acute 24 e 48 hr culture sono anche impostati. <ol><li> Raccogliere almeno 1 ml di midollo osseo in una parte superiore del tubo di sodio eparina verde. Aliquotare 0,25 ml di midollo osseo in 10 ml di mezzi RPMI completo contenente L-glutammina (20% di siero bovino fetale, 1% di penicillina / streptomicina, 1% fungizone). Coltura a 37 ° C con 5% di CO 2. </li></ol></li></ol> 2. Presentazione Preparazione e colorazione Solid Una rapida valutazione di una diapositiva rappresentante fornirà informazioni sulla qualità del raccolto prima di procedere ad ulteriori procedure come G-banding, FISH, CGH, o SKY. Alcuni laboratori preferiscono macchia solida alle cellule per la raccolta rapida e la valutazione dei cromosomi. In alternativa, un microscopio a contrasto di fase può essere utilizzata per questa analisi. I vetrini sono meglio preparati quando l&#39;umidità è circa il 50% e la temperatura ambiente (20-25 ° C). <ol><li> Centrifugare le cellule a 200 xg per 5 min a 25 ° C. Rimuovere il surnatante fino a soli 0,3-0,5 ml resti. </li><li> Dopo aver risospeso delicatamente i pellet, pipette tre gocce della sospensione di cellule da una distanza di circa 2 in su un vetrino che è inclinato di un angolo di circa 45 ° e consentire la sospensionerotolare attraverso la diapositiva. Aggiungere una grossa goccia di fissativo fresco di Carnoy alla diapositiva. </li><li> Asciugare il retro del vetrino su un tovagliolo di carta e poi sedersi il vetrino ad asciugare per almeno 10 min. La slitta deve essere completamente asciutto. </li><li> Preparare fresco Giemsa soluzione (rapporto 3:1 di Gurr Buffer e Giemsa). Porre i vetrini in un rack per colorazione. Coprire l&#39;intera diapositiva nella soluzione colorante Giemsa. Lasciare che i vetrini rimangono nella soluzione colorante per 5 min. Sciacquare i vetrini con acqua distillata, drenare, e lasciare asciugare. </li><li> Aggiungere 4 gocce di Permount e un vetrino alla diapositiva. Assicurarsi che non vi siano bolle sotto il vetrino. L&#39;eccesso Permount può essere rimosso con un tovagliolo di carta. </li><li> Analizzare le cellule con un microscopio ottico sotto 10X e 100X ingrandimento. Se le cellule in metafase sono abbondanti e ben diffusione, le diapositive rimanenti possono essere utilizzati per altre procedure sperimentali. </li></ol> 3. G-Banding Uso tripsina e Giemsa (GTG) &lt;p&gt; La tripsina è un enzima proteolitico che denatura istoni eucromatiche nelle regioni del DNA con l&#39;attività trascrizionale maggiore risultante. A seguito di Giemsa, queste regioni appariranno come bande luminose. Cromatina altamente condensato con poca o nessuna attività trascrizionale (eterocromatina) avrà un gran parte delle sue istoni protette dalla tripsina e pertanto macchia scura seguente Giemsa. È essenziale inizialmente G-band una diapositiva a monitorare le condizioni e regolare la temporizzazione tripsina se necessario per le diapositive successive. <ol><li> Aggiungere le seguenti soluzioni a 4 vaschette Coplin. Jar # 1 = 30 ml di 1x HBSS e 4 ml di tripsina 10x (0,5%) Jar # 2 = 50 ml di 1x HBSS Jar ° 3 = 45 ml di 1x HBSS e 5 ml di siero fetale bovino Jar # 4 = 50 ml di 1x HBSS </li><li> Immergere ogni diapositiva in Jar # 1 per 5 secondi, rapido risciacquo in Jar # 2, lasciare ogni diapositiva in Jar # 3 per almeno 30 secondi, rapido risciacquo in Jar # 4 poi permettono vetrini si asciughino. </li><li> Preparare fresh Giemsa colorazione Solution (rapporto 3:1 di Gurr Buffer e Giemsa). Porre i vetrini in un rack per colorazione. Coprire l&#39;intera diapositiva nella soluzione colorante Giemsa. Lasciare che i vetrini rimangono nella soluzione colorante per 5 min. </li><li> Sciacquare i vetrini in acqua distillata nello stesso ordine in cui erano macchiate. Lasciare i vetrini ad asciugare per circa 10 min. La slitta deve essere completamente asciutto. </li><li> Aggiungere 4 gocce di Permount e un vetrino alla diapositiva. Assicurarsi che non vi siano bolle sotto il vetrino. L&#39;eccesso Permount può essere rimosso con un tovagliolo di carta. </li><li> Analizzare le cellule con un microscopio ottico sotto 10X e 100X ingrandimento. Regolare la temporizzazione tripsina del resto delle diapositive sulla base dei risultati della prima slitta. </li></ol>

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Abbiamo utilizzato il presente procedimento per il cromosoma isolamento da cellule di vari organismi, tra cui vari tipi di cellule ottenute da umano, macachi Rhesus, ratti e topi 11,20-22. Il protocollo standard è disponibile, ma alcuni passaggi chiave e variabili potrebbe essere necessario regolare per questi diversi tipi di cellule. Diversi passaggi specifici sono cruciali sia nella preparazione cromosomica e G-bande per garantire la migliore qualità dei risultati. Una delle variabili che possono influenz...

Analisi cromosomica è una tecnica tradizionale utilizzata in tutto il mondo per diagnosticare instabilità cromosomica e riarrangiamenti che portano a malattie genetiche e tumori maligni 1,2,8,9. Inoltre, una risoluzione maggiore per la diagnosi e la ricerca di anomalie genetiche e costituzionali cancro acquisita può essere realizzato con la combinazione delle procedure citogenetiche classiche e metodologie citogenetiche molecolari come ibridazione in situ fluorescente (FISH), ibridazione genomica comparativa (CGH) , e cariotipo spettrale (SKY) 14,15. Più recentemente, queste tecniche sono state utilizzate per la valutazione di instabilità cromosomica associata ricerca sulle cellule staminali. Anomalie del cariotipo come aneuploidia di cellule in coltura a lungo termine embrionali (ES) e cellule staminali di diversi organismi sono stati riportati da diversi laboratori. Recenti evidenze sostiene che alcune linee cellulari sono intrinsecamente più inclini al cromosoma instabilità indipendentemente dalle condizioni di coltura. Per questo motivo, al momento di stabilire e / o il mantenimento di linee di cellule staminali umane Rhesus, mouse, o, analisi cromosomica è raccomandata come parte del processo di controllo di qualità. Molti rapporti descrivono il crescente interesse per l&#39;uso di routine e citogenetica molecolare per monitorare la stabilità cromosomica di cellule staminali e cellule maligne o vari organismi in coltura 10-13. Questi protocolli sono sempre più utilizzati dai laboratori non genetici per una rapida valutazione cromosomica delle loro cellule in coltura 13. Presentiamo nostre procedure di base per la preparazione cromosoma da vari tipi di cellule, che possono essere applicate per entrambi gli scopi e le cellule clinici e di ricerca derivati ​​da vari organismi.

<table border="0" cellpadding="0" cellspacing="0" width="662">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">KaryoMAX Colcemid</td>
			<td style="width:213px;">Gibco</td>
			<td style="width:145px;">15212-012</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS Buffer</td>
			<td>Gibco</td>
			<td>24020-117</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5% Trypsin EDTA 10x</td>
			<td>Gibco</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Permount</td>
			<td>Fisher Scientific</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buffer Tablets &#34;GURR&#34;</td>
			<td>Gibco</td>
			<td>10580-013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Geimsa Stain</td>
			<td>Ricca Chemical Company</td>
			<td>3250-16</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge 5810</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>Caledon Laboratory Chemicals</td>
			<td>6700130</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glacial Acetic Acid</td>
			<td>Krackeler Scientific Inc.&#160;</td>
			<td>11-9508-05</td>
			<td></td>
		</tr>
	</tbody>
</table>

chromosome preparation from cultured cells

Cromosoma (citogenetica) analisi è ampiamente utilizzato per il rilevamento di instabilità cromosomica. Quando seguito da G-banding e le tecniche molecolari come ibridazione in situ fluorescente (FISH), questa analisi ha la potente capacità di analizzare le singole celle per le aberrazioni che coinvolgono gli utili o le perdite di porzioni del genoma e riarrangiamenti che coinvolgono uno o più cromosomi. Negli esseri umani, anomalie cromosomiche si verificano in circa 1 ogni 160 nati vivi 1,2, il 60-80% di tutti gli aborti spontanei 3,4, il 10% dei nati morti 2,5, il 13% degli individui con malattia cardiaca congenita 6, 3-6% dei casi di infertilità 2, e in molti pazienti con ritardo dello sviluppo e difetti di nascita 7. Analisi citogenetica di malignità è abitualmente utilizzato dai ricercatori e clinici, come osservazioni di anomalie cromosomiche clonali hanno dimostrato di avere sia un significato diagnostico e prognostico 8,9.60; isolamento Cromosoma è prezioso per la terapia genica e la ricerca sulle cellule staminali degli organismi inclusi primati non umani e roditori 10-13. I cromosomi possono essere isolate da cellule di tessuti vivi, compresi i linfociti del sangue, fibroblasti cutanei, amniociti, placenta, midollo osseo, e campioni tumorali. I cromosomi sono analizzati nella fase metafase della mitosi, quando sono più condensati e quindi più visibile. Il primo passo della tecnica di isolamento cromosoma comporta la rottura delle fibre del fuso mediante incubazione con Colcemid, per evitare che le cellule di procedere alla fase successiva anafase. Le cellule sono poi trattate con una soluzione ipotonica e conservati nel loro stato gonfio di fissativo di Carnoy. Le cellule vengono poi trascinati nella vetrini e possono quindi essere utilizzati per una varietà di procedure. G-banding comprende il trattamento tripsina seguita da colorazione con Giemsa per creare luce caratteristico e bande scure. Lo stesso procedure isolare cromosomi può essere utilizzato per la preparazione di cellule per procedure quali la ibridazione in situ fluorescente (FISH), ibridazione genomica comparativa (CGH), e cariotipo spettrale (SKY) 14,15.

Spread metafase alta qualità sono essenziali per l&#39;analisi cromosomica. Un test riuscito produce cromosomi che sono ben diffuse e di adeguata morfologia cromosoma. Cromosomi Correttamente G-banda contengono la luce caratteristico e modelli di bande scure. <img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/50203/50203fig1highres.jpg" src="/files/ftp_upload/50203/50203fig1.jpg" /> Figura 1. Una diffusione cromosomico suc...

Watch this Scientific Journal Video about Chromosome Preparation From Cultured Cells at JoVE.com

Chromosome Preparation From Cultured Cells

I cromosomi possono essere isolate da cellule vive come linfociti o fibroblasti cutanei, e da organismi compresi gli esseri umani o topi. Queste preparazioni cromosomiche possono essere ulteriormente utilizzati per routine di G-banding e le procedure di citogenetica molecolare quali la ibridazione in situ fluorescente (FISH), ibridazione genomica comparativa (CGH), e cariotipo spettrale (SKY).

Cromosoma Preparazione da cellule in coltura

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

chromosome-preparation-from-cultured-cells

Research

JoVE Journal

Biology

80.7K Views.  Louisiana State University Health Science Center.  I cromosomi possono essere isolate da cellule vive come linfociti o fibroblasti cutanei, e da organismi compresi gli esseri umani o topi. Queste preparazioni cromosomiche possono essere ulteriormente utilizzati per routine di G-banding e le procedure di citogenetica molecolare quali la ibridazione in situ fluorescente (FISH), ibridazione genomica comparativa (CGH), e cariotipo spettrale (SKY). 

Video: Chromosome Preparation From Cultured Cells

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Conteggio e determinazione della vitalità delle cellule in coltura

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Ricerca

Biologia

Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells

This video demonstrates Shidham's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually scattered cells and small cell groups. This technique uses HistoGel (Thermo Scientific) with conventional laboratory equipment.
The use of cell block sections is a valuable ancillary tool for evaluation of non-gynecologic cytology. They enable the cytopathologist to study additional morphologic specimen detail including the architecture of the lesion. Most importantly, they allow for the evaluation of ancillary studies such as immunocytochemistry, in-situ hybridization tests (FISH/CISH) and in-situ polymerase chain reaction (PCR). Traditional cell block preparation techniques have mostly been applied to non-gynecologic cytology specimens, typically for body fluid effusions and fine needle aspiration biopsies.
Liquid based cervicovaginal specimens are relatively less cellular than their non-gynecologic counterparts with many individual scattered cells. Because of this, adequate cellularity within the cell block sections is difficult to achieve. In addition, the histotechnologist sectioning the block cannot visualize the level at which the cells are at the highest concentration. Therefore, it is difficult to monitor the appropriate level at which sections can be selected to be transferred to the glass slides for testing. As a result, the area of the cell block with the cells of interest may be missed, either by cutting past or not cutting deep enough. Current protocol for Shidham's method addresses these issues. Although this protocol is standardized and reported for gynecologic liquid based cytology specimens, it can also be applied to non-gynecologic specimens such as effusion fluids, FNA, brushings, cyst contents etc for improved quality of diagnostic material in cell block sections.

Shidham's method for preparation of cell blocks with AV-marker from cytology specimens containing individually scattered cells and small cell groups.

Cellule preparazione del blocco da campioni Citologia con predominanza di cellule sparse individualmente

This video demonstrates Shidham's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually ...

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

Preparazione dei cromosomi di Drosophila Zucche politene per l&#39;etichettatura di anticorpi

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Basato sulla fluorescenza Misura Store-Entry operato di calcio in cellule vive: dal Cancer Cell coltura per fibra muscolare scheletrica

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Visualizzazione del reticolo endoplasmatico sottodomini in cellule in coltura

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

Saggi Reporter massicciamente parallelo in cellule di mammifero in coltura

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Isolamento e analisi funzionale dei mitocondri da cellule in coltura e Tissue mouse

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imaging locale Ca<sup&gt; 2+</sup&gt; Segnali in cellule di mammifero coltivate

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

Un rapido aria secca cadente Cromosoma Metodo di preparazione adatto per i pesci nelle piante

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

Preparazione del cromosoma meiotico si diffonde da Mouse spermatociti

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...