Name: determination of lipid raft partitioning of fluorescently-tagged probes in living cells by fluorescence correlation spectroscopy (fcs)
Uploaded: 2012-04-06T13:00:00
Description: Watch this Scientific Journal Video about Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS at JoVE.com

Эта работа была поддержана грантом Agence Nationale-де-ла Recherche (ChoAD). Мы также благодарны Фонду ICM (институт дю Cerveau и др. де-ла-Moelle) за их финансовую поддержку.

1. Калибровка установки FCS <ol><li> Начать конфокальной микроскопии, лазеры, компьютеры, инкубатор для температуры и CO 2 контроля. </li><li> Убедитесь в том, SPAD (Single лавины фотонов Diode) на флуоресценцию и фильтр внутри SPAD хорошо подходит для вашего образца. Убедитесь, что SPAD синхронизирован?...

<ol>
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Метод ФТС представленные здесь позволяет чувствительный и быстрый анализ разделов липидного плота флуоресцентных зондов интерес в живых клетках. ФТС сочетает в себе точность локализации конфокальной микроскопии с чувствительностью одного счета фотонов. Основное различие между ФТС ...

<html><table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер по каталогу </td><td> Комментарии </td></tr><tr><td> Холерный токсин субъединицы B-Alexa 488 </td><td> Invitrogen </td><td> C-34775 </td><td> МВт (пентамера) = 57 кг / моль </td></tr><tr><td> Конфокальной микроскопии </td><td> Leica </td><td> SP5 </td><td></td></tr><tr><td> Инкубатор для температуры и СО2 контроль </td><td> Жизнь изображений услуги </td><td> Куба и коробки </td><td></td></tr><tr><td> SPAD (Single лавины фотонов Diode) </td><td> MPD (Micro фотонных устройств) </td><td> PDM серии (100 мкм чувствительная область) </td><td></td></tr><tr><td> Высокий перевал 488 нм фильтра </td><td> Semrock </td><td> 488 нм блокирование края BrightLine долго фильтр Part #ails.aspx? ID = FF01-488/LP-25 &quot;целевых =&quot; _blank &quot;&gt; FF01-488/LP-25 </td><td></td></tr><tr><td> ФТС блок детектирования </td><td> Picoquant </td><td> Picoharp 300 модулей </td><td></td></tr><tr><td> Приобретение и автокорреляции программного обеспечения </td><td> Picoquant </td><td> SymPhoTime </td><td></td></tr><tr><td> Установка программного обеспечения </td><td> OriginLab </td><td> OriginPro8 </td><td></td></tr></tbody></table>

determination of lipid raft partitioning of fluorescently-tagged probes in living cells by fluorescence correlation spectroscopy (fcs)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking1,2 but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases3.
Yet their existence is still a matter of controversy4,5. Indeed, lipid raft size has been estimated to be around 20 nm6, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized7,8. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells.
Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts9,10. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models11.
FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently12. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change12.

Watch this Scientific Journal Video about Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS at JoVE.com

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS

Техника для исследования разделов липидного плота флуоресцентных белков в мембране живой клетки описывается. Он использует различия в диффузии время белки, расположенные внутри или за пределами липидного плоты. Приобретение может осуществляться динамически в контрольных условиях или после наркомании.

Определение липидного плота разделов флуоресцентно с метками зонды в живых клетках при флуоресцентной спектроскопии корреляции (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. ...

Research

JoVE Journal

Biology

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

Measuring the Kinetics of mRNA Transcription in Single Living Cells

Измерение кинетики мРНК Транскрипция в одиночных камерах Жизнь

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy

Легко Измерение коэффициентов диффузии в EGFP-меченых белков плазматической мембраны Использование К-пространство изображения корреляционной спектроскопии

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new ...

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Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

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Флуоресценции изображений с Один-нм Точность (Fiona)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Использование CaCO 2-Cells по изучению транспорта липидов в кишечнике

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

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

Графический пользовательский интерфейс для отслеживания концентрации белка в программном сопровождении в динамических клеточных выпячиваниях

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

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Confocal микроскопии показывает клеток поверхности рецептор агрегации через спектроскопии корреляции изображений

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

Определение плазматической мембраны секционирования для периферически связанных белков

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Динамика олигомеризации рецепторов поверхности клеток в живых клетках с помощью общей внутренней флуоресценции в сочетании с анализом числа и яркости

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

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Зондирование мРНК Кинетика в пространстве и времени в Escherichia coli с помощью двухцветной одноямекулярной флуоресценции в гибридизации Situ

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...