Anyaporn Sawasdichai e Nazefah Abdul Hamid grati al governo tailandese e il Governo della Malaysia, rispettivamente per dottorato di ricerca Borse di studio. Questo lavoro è stato finanziato da un progetto Wellcome Trust sovvenzione concessa a PSJ e KG. Siamo anche grati per l&#39;Università di Bristol Fondo Bioimmagini Wolfson.

I. Preparazione per il frazionamento Questa sezione descrive la preparazione di poli-L-lisina coprioggetto microscopio rivestiti e l&#39;attaccamento di cellule prima del frazionamento. Se necessario le cellule possono essere transitoriamente transfettate con vettori di espressione delle proteine ​​prima o dopo l&#39;attaccamento. A. Preparazione del coprioggetto rivestiti di poli-L-lisina <ol><li> Preparare una soluzione di 1mg/ml poli-L-lisina in acqua distillata. </li><li> Coprioggetto cappotto pulito, con poli-L-lisina in incubazione, in nella soluzione per almeno 1 ora su una piattaforma oscillante a 22 ° C. </li><li> Lavare i coprioggetti rivestito con acqua distillata sterile due volte e seguire con un lavaggio singolo con il 96% di etanolo. </li><li> Far asciugare i vetrini rivestiti su un pezzo di carta da filtro e conservarle in un contenitore asciutto per un uso futuro (una volta asciutta possono essere impilati). </li></ol> Cellule B. Connessione a lamelle Non aderente cellule (qui usiamo cellule K562) <ol><li> Inserire una poli-L-lisina coprioggetto rivestiti in un pozzo in un 6-pozzetti con la superficie rivestita rivolta verso l&#39;alto. </li><li> Semi di cellule K562 ad una densità di 7x10 5 cellule / pozzetto in Dulbecco modificato (DMEM) supplementato con 10% siero fetale bovino (FBS) e PS (penicillina 100units/ml, 100mg/ml streptomicina). Nel caso di cellule K562 trasfezione transiente può essere eseguita con elettroporazione (0,4 centimetri cuvette elettroporazione con 1x10 7 cellule in 200μl di media a 250V / 975μF) prima di seminare le cellule. </li><li> Incubare le cellule per 24 ore a 37 ° C in 5% di CO 2. </li><li> Eliminare il medio e lavare le cellule due volte con fosfato ghiacciata soluzione salina tamponata (PBS). </li><li> Seguite con il protocollo di frazionamento subcellulare. </li></ol> Cellule aderenti (qui usiamo cellule COS-7) <ol><li> Inserire un vetrino rivestito in un pozzo in un 6-pozzetti. </li><li> Lavare due volte in PBS preriscaldata a 37 ° C. </li><li> Staccare le cellule digestione con tripsina (0,03% EDTA, 0,25% tripsina) in PBS a 37 ° C per 2 a 5 minuti. E &#39;importante non a un eccesso di digerire le cellule e per fermare la digestione non appena le cellule galleggianti prevalentemente individuali sono presenti. </li><li> Fermare la digestione seminando le cellule ad una densità di 3x10 5 cellule / pozzetto in un 6-pozzetti in DMEM supplementato con 10% FBS e PSG (penicillina 100 unità / ml, streptomicina 100ug/ml e L-glutamina 292ug/ml) , su lamelle normale. Cellule aderenti generalmente allegare anche sul coprioggetto microscopio normale, tuttavia, vetrini rivestiti di poli-L-lisina può essere utilizzato se necessario. </li><li> Incubare le cellule per 24 ore a 37 ° C e 5% di CO 2. Nel caso di cellule COS-7 trasfezione transiente può essere effettuata utilizzando Fugene 6 (Roche) se richiesto e le celle a sinistra per recuperare per altre 24 ore a 37 ° C e 5% di CO 2. </li><li> Eliminare il medio e lavare le cellule due volte con PBS freddo ghiaccio. </li><li> Seguite con il protocollo di frazionamento subcellulare. </li></ol> II. Frazionamento subcellulare Della frazione in situ viene eseguita secondo lo schema mostrato nella Figura 1 e si basa su un metodo descritto in precedenza 5 con le modifiche descritte in precedenza 7 e nel protocollo dettagliato di seguito. Si consiglia di trasferire i coprioggetti di un nuovo 6-pozzetti dopo ogni fase di frazionamento di rimuovere il vetrino prima di rimuovere il liquido. Anche se solo quattro lamelle sono necessari per l&#39;imaging, coprioggetto sono necessari ulteriori per la produzione di estrarre cellule intere, e le frazioni di matrice nucleare per western blotting se questo deve essere eseguito in parallelo. <ol><li> Preparare e filtro citoscheletro buffer (CSK): TUBI 10mm, Saccarosio 300mm, 100mM NaCl, 3mm MgCl 2, 1 mM EGTA. CSK tampone deve essere preparata il giorno di frazionamento. </li><li> Se necessario le cellule da un coprioggetto può essere utilizzato per preparare l&#39;estratto cellule intere, per western blotting con delicatezza mettendo 200ul tampone TES (1% SDS, 2mM EDTA, 20 mM Tris-HCl pH 7,4) direttamente sul coprioggetto e incubare per 1 o 2 minuti a 22 ° C. L&#39;estratto cellule intere possono quindi essere rimossi e le proteine ​​precipitate con l&#39;aggiunta di 250ul di 1M (NH 4) 2 SO 4 e incubazione su ghiaccio fino a quando gli altri campioni occidentali sono pronti. </li><li> Lavare i coprioggetti restanti con 1ml PBS freddo ghiaccio due volte a 22 ° C inclinando il 6 ben piatto due o tre volte. </li><li> Rimuovere il vetrino che rappresentano le cellule intere e trasferimento in un nuovo 6-pozzetti. Seguite con la fissazione delle cellule e l&#39;immuno-fluorescenza protocollo. </li><li> Rimuovere delicatamente la PBS dai pozzetti rimanenti. </li><li> Estrarre le proteine ​​citoplasmatiche e nucleari vagamente tenuto delicatamente mettendo 200ul tampone CSK + 0,1% (v / v) Triton X-100 direttamente su ogni coprioggetto rimanente e incubazione dighiaccio per 1 minuto. </li><li> Rimuovere l&#39;estratto citoplasmatico e vagamente tenuto nucleare e precipitare come descritto al punto 2. </li><li> Lavare i coprioggetti con ghiaccio 1ml PBS freddo due volte a 22 ° C inclinando il piatto ben 6. </li><li> Rimuovere il vetrino che rappresentano ben tenuta materiale nucleare e seguire con la fissazione delle cellule e l&#39;immuno-fluorescenza protocollo. </li><li> Rimuovere delicatamente la PBS dai pozzetti rimanenti quindi estrarre le proteine ​​strettamente tenuto delicatamente mettendo 200ul tampone CSK + 0,5% (v / v) Triton X-100 direttamente su ogni coprioggetto rimanente e incubazione in ghiaccio per 20 minuti. </li><li> Rimuovere l&#39;estratto ben tenuto e precipitare come descritto al punto 2. </li><li> Lavare i coprioggetti con ghiaccio 1ml PBS freddo due volte a 22 ° C inclinando il piatto ben 6. </li><li> Rimuovere il vetrino che rappresentano frazione cromatina e seguire con la fissazione delle cellule e l&#39;immuno-fluorescenza protocollo. </li><li> Rimuovere delicatamente la PBS dai pozzetti rimanenti poi posto 200ul CSK tampone + 100ug/ml di DNasi I rimanenti sul coprioggetto e incubare per 30 minuti a 37 ° C. </li><li> Rimuovere l&#39;estratto della cromatina e precipitare come descritto al punto 2. </li><li> Lavare i coprioggetti con ghiaccio PBS freddo due volte a 22 ° C inclinando il piatto ben 6. </li><li> Rimuovere il vetrino che rappresenta la frazione nucleare matrice e seguire con la fissazione delle cellule e l&#39;immuno-fluorescenza protocollo. </li><li> Se necessario, la proteine ​​della matrice può essere rimossa da un coprioggetto matrice aggiuntivi per western blotting utilizzando 200ul tampone TES come descritto al punto 2. </li><li> I campioni per western blot devono essere centrifugati a 13000 rpm in microcentrifuga da banco a 4 ° C ed il pellet risospeso in 40ul SDS tampone di caricamento (62,5 mm Tris-HCl pH 6.8, 2% SDS (w / v), 50 mM DTT, 10% glicerolo, 0,01% blu di bromofenolo (w / v)) prima dell&#39;analisi mediante SDS-PAGE. <img src="/files/ftp_upload/1958/1958fig1.jpg" alt="Figura 1" /> Figura 1. Una rappresentazione schematica di conservazione in situ frazionamento subcellulare. Citoscheletro buffer (CSK) si compone di TUBI 10mm, Saccarosio 300mm, 100mM NaCl, 3mm MgCl 2, 1 mM EGTA. TES tampone è costituito da 1% SDS, 2mM EDTA, 20 mM Tris-HCl pH 7,4. </li></ol> III. Fissazione delle cellule e immuno-fluorescenza <ol><li> Delicatamente aggiungere 1 ml di formaldeide al 4% / PBS per ogni coprioggetto e incubare a 4 ° C per 30 minuti. </li><li> Lavare ogni coprioggetto delicatamente 2-3 volte con 1 ml di PBS a 22 ° C. </li><li> Aggiungi 400ul di X-100/PBS Triton 0,1% per ogni coprioggetto e incubare a 22 ° C per 10 minuti. </li><li> Lavare ogni coprioggetto delicatamente 2-3 volte con 1 ml di PBS a 22 ° C. </li><li> Aggiungi 400ul del 3% di albumina sierica bovina (BSA) / PBS e incubare a 22 ° C per 20 minuti per ridurre la colorazione di fondo potenziale. </li><li> Lavare ogni coprioggetto delicatamente 2-3 volte con 1 ml di PBS a 22 ° C. </li><li> 100ul posto dell&#39;anticorpo primario in PBS direttamente sul coprioggetto e incubare a 22 ° C per 1 ora. Anticorpi primari e secondari devono essere diluiti in PBS alla concentrazione richiesta. Questo può avere bisogno di essere ottimizzato per ogni anticorpo utilizzato. </li><li> Lavare ogni coprioggetto delicatamente 2-3 volte con 1 ml di PBS a 22 ° C. </li><li> 200ul luogo di anticorpo secondario in PBS e incubare a 22 ° C al riparo dalla luce per 1 ora. </li><li> Lavare ogni coprioggetto delicatamente 2-3 volte con 1 ml di PBS a 22 ° C. </li><li> Montare ciascun vetrino coprioggetto (celle in basso) su un vetrino da microscopio in vetro con supporto Vectashield montaggio contenente DAPI (4 &#39;,6-diamidino-2-fenilindolo cloridrato). </li><li> Pulire l&#39;eccesso mezzo di montaggio di tutto il coprioggetto utilizzando un tovagliolo di carta e incubare il vetrino a 22 ° C per almeno 30 minuti al riparo dalla luce. </li><li> Fissare il coprioggetto al vetrino usando chiaro smalto acrilico. </li><li> Tenere le diapositive al riparo dalla luce prima visualizzazione al microscopio a fluorescenza. </li></ol> IV. Rappresentante risultati <img src="/files/ftp_upload/1958/1958fig2.jpg" alt="Figura 2" /> Figura 2. Frazionamento subcellulare di non transfettate cellule COS-7. Frazionato cellule COS-7 sono stati fissati con formaldeide e immunostained con α-tubulina, α-istoni H1 o α-Lamina A / C anticorpi coniugati con TRITC seguita da trattamento con mezzo di montaggio contenente DAPI . Le immagini sono state acquisite con oculare 10x e lenti dell&#39;obiettivo 63x utilizzando un microscopio confocale Leica. Nuclei delle cellule sono state visualizzate utilizzando una serie DAPI filtro mentre nelle cellule stesso campo colorate con anticorpi marcatori sono stati visualizzati utilizzando un insieme TRITC filtro. <img src="/files/ftp_upload/1958/1958fig3.jpg" alt="Figura 3" /> Figura 3. Un GFP-6E2 proteina di fusione è associato con la matrice nucleare. Frazionamento subcellulare di Cos-7 cellule che esprimono una proteina di fusione costituita da una proteina fluorescente verde (GFP) fuso con il papillomavirus umano (HPV) di tipo 6E2 proteine ​​(GFP-6E2). Nuclei delle cellule sono stati visualizzati tramite colorazione DAPI, mentre la localizzazione della GFP-6E2 nelle cellule transfettate era direttamente visualizzati tramite fluorescenza GFP. Lamina A / C è stata osservata con α-Lamina A / C anticorpi coniugati con TRITC e una serie TRITC filtro. Le immagini si fondono rivelano che alcune delle proteine ​​GFP-6E2 è associato con la matrice nucleare (in basso a destra del pannello). Le immagini sono state ottenute come descritto nella Figura 2.

<ol>
	<li>Donaldson, M. M., Boner, W., Morgan, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopBP1+regulates+human+papillomavirus+type+16+E2+interaction+with+chromatin.">TopBP1 regulates human papillomavirus type 16 E2 interaction with chromatin.</a> J Virol. 81, 4338-4342 (2007).</li><li>Javed, A., Guo, B., Hiebert, S., Choi, J. Y., Green, J., Zhao, S. C., Osborne, M. A., Stifani, S., Stein, J. L., Lian, J. B., van Wijnen, A. J., Stein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Groucho/TLE/R-esp+proteins+associate+with+the+nuclear+matrix+and+repress+RUNX+(CBF(alpha)/AML/PEBP2(alpha))+dependent+activation+of+tissue-specific+gene+transcription.">Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBF(alpha)/AML/PEBP2(alpha)) dependent activation of tissue-specific gene transcription.</a> J Cell Sci. 113, 2221-2231 (2000).</li><li>Kowalczyk, A. M., Roeder, G. E., Green, K., Stephens, D. J., Gaston, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+induction+or+inhibition+of+apoptosis+by+HPV+proteins.">Measuring the induction or inhibition of apoptosis by HPV proteins.</a> Methods Mol. Med. 119, 419-432 (2005).</li><li>McLarren, K. W., Theriault, F. M., Stifani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+with+the+nuclear+matrix+and+interaction+with+Groucho+and+RUNX+proteins+regulate+the+transcription+repression+activity+of+the+basic+helix+loop+helix+factor+Hes1.">Association with the nuclear matrix and interaction with Groucho and RUNX proteins regulate the transcription repression activity of the basic helix loop helix factor Hes1.</a> J Biol. Chem. 276, 1578-1584 (2001).</li><li>McNeil, S., Guo, B., Stein, J. L., Lian, J. B., Bushmeyer, S., Seto, E., Atchison, M. L., Penman, S., van Wijnen, A. J., Stein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+the+YY1+transcription+factor+to+the+nucleolus+and+the+nuclear+matrix+in+situ:+the+C-terminus+is+a+principal+determinant+for+nuclear+trafficking.">Targeting of the YY1 transcription factor to the nucleolus and the nuclear matrix in situ: the C-terminus is a principal determinant for nuclear trafficking.</a> J Cell Biochem. 68, 500-510 (1998).</li><li>Melan, M. A., Sluder, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redistribution+and+differential+extraction+of+soluble+proteins+in+permeabilized+cultured+cells.+Implications+for+immunofluorescence+microscopy.">Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Implications for immunofluorescence microscopy.</a> J Cell Sci. 101, 731-743 (1992).</li><li>Zou, N., Lin, B. Y., Duan, F., Lee, K. Y., Jin, G., Guan, R., Yao, G., Lefkowitz, E. J., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hinge+of+the+human+papillomavirus+type+11+E2+protein+contains+major+determinants+for+nuclear+localization+and+nuclear+matrix+association.">The hinge of the human papillomavirus type 11 E2 protein contains major determinants for nuclear localization and nuclear matrix association.</a> J. Virol. 74, 3761-3770 (2000).</li></ol>

Nessun conflitto di interessi dichiarati.

Problemi comuni e suggerimenti: Tutti o la maggior parte delle cellule sono persi durante il lavaggio. Durante le fasi di lavaggio deve essere aggiunto lentamente al lato del 6-pozzetti evitando il coprioggetto. Allo stesso modo i liquidi devono essere rimossi con attenzione inclinando la piastra e lentamente pipettaggio l&#39;eccesso di liquido. L&#39;adesione cellulare può essere aumentata mediante coprioggetto rivestiti di poli-L-lisina. DNA genomico non è del ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>BSA</td>
<td>Chemical</td>
<td>Sigma</td>
<td>A9647-100G</td>
<td>&nbsp;</td>
<tr>
<td>DNase I</td>
<td>Chemical</td>
<td>Sigma</td>
<td>DN25-1G</td>
<td>&nbsp;</td>
<tr>
<td>poly-L-Lysine</td>
<td>Chemical</td>
<td>Sigma</td>
<td>P-8920</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>Chemical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>EGTA</td>
<td>Chemical</td>
<td>Sigma</td>
<td>E4378-25G </td>
<td>&nbsp;</td>
<tr>
<td>Fomaldehyde</td>
<td>Chemical</td>
<td>BDH</td>
<td>284216N</td>
<td>&nbsp;</td>
<tr>
<td>PIPES</td>
<td>Chemical</td>
<td>Sigma</td>
<td>P-6757</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>Chemical</td>
<td>Fisher Scientific</td>
<td>S/8600/53</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Chemical</td>
<td>Sigma</td>
<td>T-6876</td>
<td>&nbsp;</td>
<tr>
<td>Mounting Medium with DAPI</td>
<td>Chemical</td>
<td>Vector Laboratories</td>
<td>H-1200</td>
<td>&nbsp;</td>
<tr>
<td>Fugene 6 Transfection Reagent</td>
<td>Chemical</td>
<td>Roche </td>
<td>11814443001</td>
<td>&nbsp;</td>
<tr>
<td>Histone H1</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-8030</td>
<td>&nbsp;</td>
<tr>
<td>Lamin A/C</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-20681</td>
<td>&nbsp;</td>
<tr>
<td>Tubulin-&alpha;</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-32293</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

in situ subcellular fractionation of adherent and non-adherent mammalian cells

Funzione della proteina è intimamente unita alla localizzazione delle proteine. Sebbene alcune proteine ​​sono limitate a una posizione specifica o compartimento subcellulare, molte proteine ​​sono presenti come una popolazione liberamente diffondere in libero scambio con una sotto-popolazione che è strettamente associata a un determinato dominio o struttura subcellulare. Frazionamento subcellulare in situ permette la visualizzazione di compartimentalizzazione delle proteine ​​e può anche rivelare le proteine ​​sotto-popolazioni che si localizzano a strutture specifiche. Per esempio, la rimozione di proteine ​​solubili citoplasmatici e vagamente tenuto proteine ​​nucleari in grado di rivelare l&#39;associazione stabile di alcuni fattori di trascrizione con cromatina. Successiva digestione del DNA possono in alcuni casi rivelano associazione con la rete di proteine ​​e RNA che viene collettivamente definito il nucleare patibolo o matrice nucleare. Qui si descrivono le operazioni necessarie durante il frazionamento in situ di cellule di mammifero aderenti e non aderenti a lamelle microscopio. Visualizzazione di proteine ​​può essere ottenuto utilizzando anticorpi specifici o proteine ​​di fusione fluorescenti e microscopia a fluorescenza. Anticorpi e / o coloranti fluorescenti che agiscono come marcatori per specifici comparti o strutture consentono la localizzazione delle proteine ​​da mappare nel dettaglio. Frazionamento in situ può anche essere combinato con western blotting per confrontare la quantità di proteine ​​presenti in ogni frazione. Questo semplice approccio biochimico in grado di rivelare le associazioni che altrimenti rimarrebbero inosservati.

Watch this Scientific Journal Video about In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells at JoVE.com

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ frazionamento subcellulare delle cellule di mammifero su vetrini microscopio permette la visualizzazione della localizzazione delle proteine.

In situ Frazionamento subcellulare di cellule di mammifero aderenti e non aderenti

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

in-situ-subcellular-fractionation-adherent-non-adherent-mammalian

Research

JoVE Journal

Biology

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

15.7K Views.  University of Bristol. In situ frazionamento subcellulare delle cellule di mammifero su vetrini microscopio permette la visualizzazione della localizzazione delle proteine.

Video: In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinizing e Trapianto cellule di mammifero

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Ricerca

Biologia

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

EsiRNA MISSIONE per lo screening RNAi in cellule di mammifero

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagenesi e Analisi funzionale dei canali ionici Heterologously Espresso in cellule di mammifero

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Clonogenica saggio: cellule aderenti

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Analisi della posizione ciclo cellulare nelle cellule di mammifero

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

Visualizzazione di mRNA reticolo endoplasmatico localizzate in cellule di mammifero

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

Polisoma Frazionamento e analisi dei mammiferi Translatomes su scala Genome-wide

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

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 ...

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 Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Un&#39;interfaccia utente grafica per il monitoraggio assistito della concentrazione di proteine ​​in protrusioni cellulari dinamiche

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...