Name: live cell imaging of f-actin dynamics via fluorescent speckle microscopy (fsm)
Uploaded: 2009-08-05T19:00:00
Description: Watch this Scientific Journal Video about Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM at JoVE.com

De ontwikkeling van qFSM wordt gefinancierd door de NIH subsidie ​​U01 GM06230.

Deel 1: Het verkrijgen van uw fluorescent gelabelde actine voor FSM Materialen die nodig zijn: gezuiverd actine, fluorofoor (Alexa, X-rhodamine aanbevolen). G-buffer, ultracentrifuge <ol><li> Een belangrijk onderdeel voor het verwerven van een goede FSM films is de juiste etikettering van actine (of andere cytoskeleteiwitten) met de fluorofoor van uw keuze. Wij raden u aan fluoroforen van Alexa (488, 568 golflengten) en X-rhodamine succinimidylester derivaten die lysineresiduen rich...

<ol>
	<li>Waterman-Storer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+speckle+microscopy+(FSM)+of+microtubules+and+actin+in+living+cells.">Fluorescent speckle microscopy (FSM) of microtubules and actin in living cells.</a> Curr Protoc Cell Biol. Chapter 4, Unit 4.10-Unit 4.10 (2002).</li><li>Ponti, A., Machacek, M., Gupton, S. L., Waterman-Storer, C. M., Danuser, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+actin+networks+drive+the+protrusion+of+migrating+cells.">Two distinct actin networks drive the protrusion of migrating cells.</a> Science. 305, 1782-1786 (2004).</li><li>Zhang, X. F., Schaefer, A. W., Burnette, D. T., Schoonderwoert, V. T., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho-dependent+contractile+responses+in+the+neuronal+growth+cone+are+independent+of+classical+peripheral+retrograde+actin+flow.">Rho-dependent contractile responses in the neuronal growth cone are independent of classical peripheral retrograde actin flow.</a> Neuron. 40, 931-944 (2003).</li><li>Salmon, W. C., Adams, M. C., Waterman-Storer, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-wavelength+fluorescent+speckle+microscopy+reveals+coupling+of+microtubule+and+actin+movements+in+migrating+cells.">Dual-wavelength fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells.</a> J Cell Biol. 158, 31-37 (2002).</li><li>Danuser, G., Waterman-Storer, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fluorescent+speckle+microscopy+of+cytoskeleton+dynamics.">Quantitative fluorescent speckle microscopy of cytoskeleton dynamics.</a> Annu Rev Biophys Biomol Struct. 35, 361-387 (2006).</li></ol>

Een aantal belangrijke factoren van cruciaal belang zijn voor het succesvol behalen van spikkel beelden, hoe goed spikkels beginnen en eindigen met de kwaliteit van uw fluorescent gelabelde actine. Een hoge verhouding tussen de etikettering van kleurstof tot actine monomeren, die rond 0,4 tot 0,7 zal ervoor zorgen dat spikkels zal verschijnen helder en discreet, terwijl de gelabelde eiwit zelf moet oplosbaar en vrij van aggregaten. Even belangrijk is de micro-injectie procedure. Om ervoor te zorgen normale cel homeostas...

Injectie buffer (IB) 50 mM kalium glutamaat 0,5 M MgCl2 (bijlage 2A) Sla tot 2 jaar bij -20 ° C &nbsp; G-buffer 2 mM Tris ⋅ Cl, pH 8.0 (bijlage 2A) 0,2 mM CaCl2 (bijlage 2A) Voeg vlak voor gebruik: 0,2 mM ATP (zie recept voor 100 mM) 0,5 mM 2-mercaptoethanol Sla tot 1 dag bij 4 ° C

live cell imaging of f-actin dynamics via fluorescent speckle microscopy (fsm)

In dit protocol beschrijven we het gebruik van TL-Speckle Microscopy (FSM) om hoge-resolutie afbeeldingen van actine dynamiek vast te leggen in PtK1 cellen. Een uniek voordeel van FSM is de mogelijkheid om de beweging en de omzet kinetiek (montage / demontage) van de F-actine-netwerk vast te leggen in levende cellen. Deze techniek is vooral handig bij de afleiding van kwantitatieve metingen van F-actine dynamiek wanneer gecombineerd met computer vision software (qFSM). We beschrijven de selectie, micro-injectie en visualisatie van fluorescerende probes actine in levende cellen. Belangrijk is dat soortgelijke procedures zijn van toepassing op het visualiseren van andere macomolecular assemblages. FSM is aangetoond voor microtubuli, intermediaire filamenten, en hechting complexen.

Watch this Scientific Journal Video about Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM at JoVE.com

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selectie, micro-injectie, en de beeldvorming van fluorescent-gelabeld F-actine via fluorescentie microscopie spikkel (FSM).

Live cell imaging van de F-actine Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

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Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

Actine Co-Sedimentatie Assay, want de analyse van eiwit binding aan F-actine

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

Montage, laden, en uitlijning van een Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

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Fluorescentie-gebaseerde waardering van Store-bediende Calcium Entry in levende cellen: van Cultured Cancer Cell op 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 ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

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Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

Real-time beeldvorming van Plant Cell Surface Dynamics met variabele hoek epifluorescentiemicroscopie

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

Driedimensionale Super resolutie microscopie van de F-actine filamenten door interferometrische Photoactivated Localization Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

Exploitatie van de levende Imaging te Track kernen tijdens Myoblast differentiatie en Fusion

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Onderzoek van de kern dynamiek van de mitotische en Meiotische splijtstof gist door fluorescentie-Live-cel microscopie

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

Live Cell Imaging om de dynamiek van de metafase timing en het lot van de cel na de mitotische spindel perturbations te beoordelen

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, ...