Anyaporn Sawasdichai et Nazefah Abdul Hamid sommes reconnaissants envers le gouvernement royal thaïlandais et le Gouvernement de la Malaisie, respectivement, pour un doctorat Bourses. Ce travail a été financé par une subvention du Wellcome Trust de projet attribué à PSJ et KG. Nous sommes également reconnaissants à l&#39;Université de Bristol Facilité Bioimaging Wolfson.

I. Préparation pour le fractionnement Cette section décrit la préparation de poly-L-Lysine lamelles de microscope enduit et la fixation des cellules avant de fractionnement. Si nécessaire, les cellules peuvent être transitoirement transfectées avec des vecteurs d&#39;expression de protéines avant ou après la fixation. A. Préparation de poly-L-Lysine lamelles revêtement <ol><li> Préparer une solution de 1mg/ml poly-L-lysine dans l&#39;eau distillée. </li><li> Lamelles pelage propre à la poly-L-Lysine en les incubant dans la solution pendant au moins 1 heure sur une plate-forme à bascule à 22 ° C. </li><li> Lavez les lamelles recouverte d&#39;eau distillée stérile à deux reprises et suivi avec un seul lavage avec de l&#39;éthanol à 96%. </li><li> Sécher à l&#39;air les lamelles couché sur un morceau de papier filtre et les conserver dans un récipient sec pour une utilisation future (une fois secs, ils peuvent être empilés). </li></ol> Cellules B. Attachement à lamelles Cellules non adhérentes (ici nous utilisons K562) <ol><li> Placer une lamelle de poly-L-Lysine couché dans un puits dans une plaque à 6 puits avec la surface revêtue vers le haut. </li><li> Graine K562 à une densité de 7x10 5 cellules / puits dans du milieu Dulbecco Modified Eagles (DMEM) supplémenté avec 10% sérum de veau fœtal (FBS) et PS (pénicilline 100units/ml, 100mg/ml streptomycine). Dans le cas des cellules K562 transfection transitoire peut être effectuée en utilisant l&#39;électroporation (0.4cm cuvettes d&#39;électroporation avec 1x10 7 cellules dans 200 ul de médias à 250V / 975μF) avant l&#39;ensemencement des cellules. </li><li> Incuber les cellules pendant 24 heures à 37 ° C dans 5% de CO 2. </li><li> Décanter le milieu et laver les cellules deux fois avec du phosphate glacée saline tamponnée (PBS). </li><li> Suivez avec le protocole de fractionnement subcellulaire. </li></ol> Les cellules adhérentes (ici nous utilisons cellules Cos-7) <ol><li> Placer une lamelle non couché dans un puits dans une plaque à 6 puits. </li><li> Laver deux fois dans du PBS préchauffé à 37 ° C. </li><li> Détacher les cellules par digestion par la trypsine (0,03% d&#39;EDTA, 0,25% de trypsine) dans du PBS à 37 ° C pendant 2 à 5 minutes. Il est important de ne pas trop assimiler les cellules et d&#39;arrêter la digestion dès majoritairement individuels cellules flottantes sont présents. </li><li> Arrêter la digestion par l&#39;ensemencement des cellules à une densité de 3x10 5 cellules / puits dans une plaque de 6 puits dans du DMEM supplémenté avec 10% de FBS et le PSG (pénicilline 100 unités / ml, streptomycine 100ug/ml et L-glutamine 292ug/ml) , sur des lamelles normale. Les cellules adhérentes attachent généralement bien sur des lamelles de microscope normal, cependant, la poly-L-Lysine lamelles revêtement peut être utilisé si nécessaire. </li><li> Incuber les cellules pendant 24 heures à 37 ° C et 5% de CO 2. Dans le cas des cellules Cos-7 transfection transitoire peut être effectuée en utilisant Fugene 6 (Roche) si nécessaire et les cellules de gauche à récupérer pour une période supplémentaire de 24 heures à 37 ° C et 5% de CO 2. </li><li> Décanter le milieu et laver les cellules deux fois avec PBS glacé. </li><li> Suivez avec le protocole de fractionnement subcellulaire. </li></ol> II. Fractionnement subcellulaire Dans fraction in situ est réalisée selon le schéma de la figure 1 et est basé sur une méthode décrite précédemment 5 avec des modifications décrites précédemment 7 et dans le protocole détaillé ci-dessous. Il est recommandé de transférer les lamelles d&#39;une nouvelle plaque de 6 puits après chaque étape de fractionnement enlever la lamelle avant d&#39;enlever le liquide. Bien que seulement quatre lamelles sont requises pour l&#39;imagerie, des lamelles supplémentaires sont nécessaires pour produire l&#39;extrait de cellules entières et des fractions de la matrice nucléaire pour western blot, si ce n&#39;est d&#39;être réalisées en parallèle. <ol><li> Préparer et filtre cytosquelette tampon (CSK): TUYAUX 10 mM, saccharose 300mm, 100mm de NaCl, 3mm MgCl2, 1 mM EGTA. Tampon CSK doit être fraîchement préparée le jour de fractionnement. </li><li> Si nécessaire les cellules d&#39;une lamelle peut être utilisé pour préparer extrait cellulaire entier pour western blot en plaçant délicatement 200ul tampon TES (1% SDS, 2 mM EDTA, 20 mM Tris-HCl pH 7,4) directement sur la lamelle et incuber pendant 1 ou 2 minutes à 22 ° C. L&#39;extrait de cellules entières peuvent ensuite être retirés et les protéines précipitées en ajoutant 250ul de 1M (NH 4) 2 SO 4 et incubation sur la glace jusqu&#39;à ce que les autres échantillons occidentaux sont prêts. </li><li> Lavez les lamelles restantes avec 1 ml PBS glacé à deux reprises à 22 ° C en inclinant la plaque de 6 bien deux ou trois fois. </li><li> Retirer la lamelle représentant des cellules entières et le transfert à un nouveau 6-même assiette. Suivez avec la fixation des cellules et l&#39;immuno-fluorescence protocole. </li><li> Retirez délicatement le PBS dans les puits restants. </li><li> Extraire les protéines cytoplasmiques et nucléaires lâchement en plaçant délicatement 200ul CSK tampon + 0,1% (V / V) de Triton X-100 directement sur chaque lamelle restant et l&#39;incubation desla glace pendant 1 minute. </li><li> Retirer l&#39;extrait cytoplasmique et lâchement nucléaire et précipiter comme décrit dans l&#39;étape 2. </li><li> Lavez les lamelles avec 1 ml PBS glacé à deux reprises à 22 ° C en inclinant le bien-six plaques. </li><li> Retirer la lamelle représentant étroitement tenu des matières nucléaires et de suivre avec la fixation des cellules et l&#39;immuno-fluorescence protocole. </li><li> Retirez délicatement le PBS dans les puits restants, puis extraire les protéines étroitement tenues en plaçant délicatement 200ul CSK tampon + 0,5% (V / V) de Triton X-100 directement sur chaque lamelle restant et l&#39;incubation sur la glace pendant 20 minutes. </li><li> Retirer l&#39;extrait bien eu lieu et le précipité comme décrit dans l&#39;étape 2. </li><li> Lavez les lamelles avec 1 ml PBS glacé à deux reprises à 22 ° C en inclinant le bien-six plaques. </li><li> Retirer la lamelle représentant fraction de la chromatine et suivi avec la fixation des cellules et l&#39;immuno-fluorescence protocole. </li><li> Retirez délicatement le PBS dans les puits restants ont alors lieu 200ul CSK tampon + 100ug/ml de DNase I sur les lamelles restantes et incuber pendant 30 minutes à 37 ° C. </li><li> Retirer l&#39;extrait de la chromatine et précipitée comme décrit dans l&#39;étape 2. </li><li> Lavez les lamelles de glace PBS froid à deux reprises à 22 ° C en inclinant le bien-six plaques. </li><li> Retirer la lamelle représentant la fraction de la matrice nucléaire et de suivre avec la fixation des cellules et l&#39;immuno-fluorescence protocole. </li><li> Si nécessaire protéines de la matrice peut être retiré de la matrice une lamelle supplémentaire pour Western blot en utilisant 200ul tampon TES comme décrit dans l&#39;étape 2. </li><li> Les échantillons pour test western blot doit être centrifugé à 13000 rpm dans une microcentrifugeuse de paillasse à 4 ° C et les culots remis en suspension dans 40ul SDS tampon de chargement (62,5 mm de Tris-HCl pH 6,8, SDS 2% (p / v), 50 mM de DTT, glycérol à 10%, 0,01% bleu de bromophénol (p / v)) avant l&#39;analyse par SDS-PAGE. <img src="/files/ftp_upload/1958/1958fig1.jpg" alt="Figure 1" /> Figure 1. Une représentation schématique d&#39;au fractionnement subcellulaire situ. Cytosquelette tampon (CSK) est constitué de 10 mM PIPES, saccharose 300mm, 100mm de NaCl, 3mm MgCl2, 1 mM EGTA. Tampon TES se compose de 1% de SDS, 2 mM EDTA, 20 mM Tris-HCl pH 7,4. </li></ol> III. Fixation de cellules et d&#39;immuno-fluorescence <ol><li> Ajouter délicatement 1ml de formaldéhyde à 4% / PBS dans chaque lamelle et incuber à 4 ° C pendant 30 minutes. </li><li> Laver chaque lamelle doucement 2-3 fois avec 1ml de PBS à 22 ° C. </li><li> Ajouter 400ul d&#39;X-100/PBS Triton de 0,1% à chaque lamelle et incuber à 22 ° C pendant 10 minutes. </li><li> Laver chaque lamelle doucement 2-3 fois avec 1ml de PBS à 22 ° C. </li><li> Ajouter 400ul de 3% albumine sérique bovine (BSA) / PBS et incuber à 22 ° C pendant 20 minutes pour réduire les taches de fond potentiels. </li><li> Laver chaque lamelle doucement 2-3 fois avec 1ml de PBS à 22 ° C. </li><li> 100ul Lieu de l&#39;anticorps primaire dans le PBS directement sur la lamelle et incuber à 22 ° C pendant 1 heure. Les anticorps primaires et secondaires doivent être dilués dans du PBS à la concentration requise. Ce peut-être besoin d&#39;être optimisé pour chaque anticorps utilisé. </li><li> Laver chaque lamelle doucement 2-3 fois avec 1ml de PBS à 22 ° C. </li><li> Lieu de 200ul anticorps secondaire dans le PBS et incuber à 22 ° C loin de la lumière pendant 1 heure. </li><li> Laver chaque lamelle doucement 2-3 fois avec 1ml de PBS à 22 ° C. </li><li> Montez chaque lamelle (cellules vers le bas) sur une lame de microscope en verre en utilisant un milieu de montage Vectashield contenant du DAPI (4 &#39;,6-diamidino-2-phénylindole chlorhydrate). </li><li> Essuyer le milieu de montage à travers la lamelle à l&#39;aide d&#39;une serviette en papier et incuber la lame à 22 ° C pendant au moins 30 minutes de lumière. </li><li> Fixer la lamelle à l&#39;aide de la lame de verre clair vernis acrylique. </li><li> Gardez les diapositives abri de la lumière avant la visualisation par microscopie à fluorescence. </li></ol> IV. Les résultats représentatifs <img src="/files/ftp_upload/1958/1958fig2.jpg" alt="Figure 2" /> Figure 2. Fractionnement subcellulaire de non-transfectées cellules COS-7. Fractionnée cellules Cos-7 ont été fixées avec du formaldéhyde et immunocolorés utilisant α-tubuline, α-histone H1 ou α-lamine A anticorps / C conjugué à la TRITC suivi par un traitement avec le milieu de montage contenant DAPI . Les images ont été acquises avec lentille oculaire 10x et 63x objectif en utilisant un microscope confocal Leica. Noyaux des cellules ont été visualisées en utilisant un ensemble DAPI filtre alors que dans le même domaine cellules colorées avec des anticorps marqueurs ont été visualisées en utilisant un ensemble TRITC filtre. <img src="/files/ftp_upload/1958/1958fig3.jpg" alt="Figure 3" /> Figure 3. Une protéine de fusion GFP-6E2 est associée à la matrice nucléaire. Fractionnement subcellulaire des cellules Cos-7 exprimant une protéine de fusion constituée de la protéine fluorescente verte (GFP) fusionnée à virus du papillome humain (VPH) de type 6E2 protein (GFP-6E2). Noyaux des cellules ont été visualisées par l&#39;intermédiaire coloration DAPI alors la localisation de la GFP-6E2 dans les cellules transfectées a été visualisé directement via fluorescence de la GFP. Lamine A / C a été visualisée en utilisant α-lamine A anticorps / C conjugué à la TRITC et un ensemble TRITC filtre. Les images révèlent que certains fusionnent de la protéine GFP-6E2 est associée à la matrice nucléaire (en bas à droite). Les images ont été obtenues comme décrit dans la figure 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>

Les problèmes courants et suggestions: Tous ou la plupart des cellules sont perdues pendant le lavage. Pendant les étapes liquides de lavage doit être ajouté lentement sur le côté de la plaque de 6 puits en évitant la lamelle. De même les liquides doivent être enlevés par attentivement inclinant la plaque et lentement pipetage hors excès de liquide. L&#39;adhésion cellulaire peut être augmentée en utilisant la poly-L-Lysine lamelles enrobées. L&#39;AD...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
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in situ subcellular fractionation of adherent and non-adherent mammalian cells

La fonction des protéines est intimement couplée à la localisation des protéines. Bien que certaines protéines sont limités à un endroit précis ou un compartiment subcellulaire, de nombreuses protéines sont présentes en tant que population librement diffusant dans le libre-échange avec une sous-population qui est étroitement associé à un domaine particulier ou d&#39;une structure subcellulaire. En fractionnement subcellulaire in situ permet la visualisation de compartimentation de protéines et peut aussi révéler des protéines sous-populations qui localisent à des structures spécifiques. Par exemple, la suppression des protéines solubles dans le cytoplasme et lâchement protéines nucléaires peut révéler l&#39;association stable de certains facteurs de transcription à la chromatine. Après la digestion de l&#39;ADN peut dans certains cas révéler association avec le réseau de protéines et d&#39;ARN qui est collectivement appelés le nucléaire échafaudage ou de la matrice nucléaire. Nous décrivons ici les étapes nécessaires au cours des fractionnement in situ de adhérentes et cellules non adhérentes de mammifères sur des lamelles de microscope. La visualisation des protéines peut être réalisée en utilisant des anticorps spécifiques ou des protéines de fusion fluorescentes et la microscopie à fluorescence. Les anticorps et / ou des colorants fluorescents qui agissent comme des marqueurs pour les compartiments spécifiques ou des structures permettant la localisation des protéines à être cartographié en détail. En fractionnement in situ peut également être combiné avec Western blot pour comparer les quantités de protéines présentes dans chaque fraction. Cette approche simple biochimiques peuvent révéler des associations qui seraient autrement inaperçus.

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

En fractionnement subcellulaire in situ des cellules de mammifères sur des lamelles de microscope permet la visualisation de la localisation des protéines.

En fractionnement subcellulaire situ des adhérentes et non-adhérents des cellules de mammifères

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

15.6K vues.  University of Bristol. En fractionnement subcellulaire in situ des cellules de mammifères sur des lamelles de microscope permet la visualisation de la localisation des protéines.

Vidéo: 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.

Trypsinisation et Repiquage des cellules de mammifères

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

Recherche

Biologie

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 MISSION pour le dépistage de l&#39;ARNi dans les cellules de mammifères

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.

Mutagenèse et analyse fonctionnelle des canaux ioniques hétérologue exprimée dans les cellules de mammifères

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.

Test clonogénique: cellules adhérentes

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.

Analyse de la situation du cycle cellulaire dans les cellules de mammifères

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

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.

Polysome Fractionnement et analyse des mammifères Translatomes sur une échelle de l&#39;ensemble du génome

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.

Mesure des niveaux Heme synthèse dans des cellules de mammifères

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.

Imagerie Ca locale<sup&gt; 2+</sup&gt; Signaux de cellules de mammifères cultivées

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.

Une interface utilisateur graphique pour le suivi assisté par logiciel de la concentration de protéines dans les protrusions cellulaires dynamiques

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

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

Coloration immunofluorescente in situ de l&#39;autophagie dans les cellules souches musculaires

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...