Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Ofir Klein; Amit Roded; Koret Hirschberg; Mitsunori Fukuda; Stephen J. Galli; Ronit Sagi-Eisenberg

doi:10.3791/57936

Imaging-FITC-Dextran als Reporter für regulierte Exozytose

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Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Imaging-FITC-Dextran als Reporter für regulierte Exozytose

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04:50 min

June 20, 2018

DOI: 10.3791/57936-v

Ofir Klein¹, Amit Roded¹, Koret Hirschberg², Mitsunori Fukuda³, Stephen J. Galli⁴, Ronit Sagi-Eisenberg¹

¹Department of Cell and Developmental Biology, Sackler Faculty of Medicine,Tel Aviv University, ²Department of Pathology, Sackler Faculty of Medicine,Tel Aviv University, ³Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences,Tohoku University, ⁴Departments of Pathology and of Microbiology and Immunology and Sean N. Parker Center for Allergy and Asthma Research, School of Medicine,Stanford University

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Overview

This article details a method for live cell imaging of regulated exocytosis using FITC-dextran as a reporter. The technique allows researchers to distinguish between different modes of exocytosis in living cells with minimal perturbation.

Key Study Components

Area of Science

Cell Biology
Neuroscience
Exocytosis Research

Background

Regulated exocytosis is crucial for various cellular functions.
Understanding exocytosis can provide insights into immune responses.
Current methods may require genetic manipulation, complicating studies.
This method offers a simpler alternative for live cell imaging.

Purpose of Study

To develop a straightforward method for studying exocytosis in live cells.
To differentiate between various modes of regulated exocytosis.
To apply this technique to different secretory cells.

Methods Used

Preparation of a stock solution of 20x Tyrode's buffer.
Mixing FITC-dextran powder with culture media.
Filtering the solution with a cellulose acetate syringe filter.
Live cell imaging to observe exocytosis.

Main Results

The method successfully visualizes regulated exocytosis in living cells.
Different modes of exocytosis can be distinguished effectively.
Applicable to mast cells, neutrophils, and eosinophils.
Minimal perturbation to cells allows for accurate observations.

Conclusions

This method provides a valuable tool for studying exocytosis.
It enhances understanding of cellular secretion mechanisms.
Future applications may extend to various secretory cell types.

Frequently Asked Questions

What is regulated exocytosis?

Regulated exocytosis is a process where cells release substances in response to specific signals.

How does FITC-dextran work in this method?

FITC-dextran accumulates in lysosome-related organelles and serves as a reporter for imaging exocytosis.

Can this method be used for all cell types?

While primarily tested on mast cells, it can also be applied to neutrophils and eosinophils.

What are the advantages of this imaging technique?

The technique is simple, requires minimal cell perturbation, and allows for live cell observation.

What is the first step in the method?

The first step is preparing a stock solution of 20x Tyrode's buffer.

Is genetic manipulation required for this method?

No, this method does not require genetic manipulation, making it easier to use.

Hier zeigen wir eine Methode für das live Cell Imaging von regulierten Exozytose. Diese Methode nutzt FITC-Dextran, die reichert sich in Lysosomen-bezogene Organellen, als Reporter. Diese einfache Methode ermöglicht auch die Unterscheidung zwischen verschiedenen Modi der regulierten Exozytose in Zellen, die schwer zu genetisch zu manipulieren sind.

Diese Methode kann dazu beitragen, Schlüsselfragen in der Erforschung der regulierten Exozytose zu beantworten, indem sie es den Forschern ermöglicht, zwischen den verschiedenen Arten der Exozytose in der lebenden Zelle zu unterscheiden. Der Hauptvorteil dieser Technik besteht darin, dass sie einfach ist und nur minimale Störungen für die Zellen erfordert. Obwohl diese Methode Einblicke in die Exozytose von Mastzellen geben kann, kann sie auch auf andere sekretorische Zellen wie Neutrophile und Eosinophile angewendet werden.

Bereiten Sie zunächst eine Stammlösung von 20x Tyrode-Puffer gemäß dem Textprotokoll vor. Mischen Sie anschließend drei Milligramm FITC-Dextran-Pulver mit drei Millilitern Nährmedium. Filtrieren Sie das gelöste FITC-Dextan mit einer Celluloseacetat-Spritzenfiltereinheit.

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