Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Luo Jia; Karen  L. Edelblum

doi:10.3791/59853

Imagem latente intravital de linfócitos intraepithelial no intestino pequeno murine

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Imagem latente intravital de linfócitos intraepithelial no intestino pequeno murine

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08:00 min

June 24, 2019

DOI: 10.3791/59853-v

Luo Jia¹, Karen L. Edelblum¹

¹Center for Immunity and Inflammation, Department of Pathology, Immunology and Laboratory Medicine,Rutgers New Jersey Medical School

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Overview

This article describes a method for visualizing GFP-labeled 纬未 IELs through intravital imaging of the murine small intestine using inverted spinning disk confocal microscopy. This technique allows for the tracking of live cells within the mucosa for up to 4 hours, facilitating the investigation of various intestinal immune-epithelial interactions.

Key Study Components

Area of Science

Neuroscience
Immunology
Cell Biology

Background

Intravital microscopy provides insights into cellular interactions within the intestinal epithelium.
The technique allows for high-speed, high-resolution imaging in real-time.
Live imaging can enhance understanding of mucosal immunity and epithelial biology.
Challenges include mastering surgical techniques and optimal mouse positioning.

Purpose of Study

To visualize live immune cells in the intestinal mucosa.
To investigate immune-epithelial interactions in real-time.
To enhance understanding of cellular dynamics in mucosal immunity.

Methods Used

Intravital imaging using inverted spinning disk confocal microscopy.
Injection of Hoechst 33342 Dye Solution into the retro-orbital venous sinus.
Monitoring of live cells for up to 4 hours.
Use of transgenic mice expressing enhanced green fluorescent protein (EGFP).

Main Results

Successful visualization of 纬未 IELs in the murine small intestine.
Real-time tracking of cellular interactions within the mucosa.
Insights into the dynamics of immune cell interactions.
Demonstration of the technique's potential for studying mucosal immunity.

Conclusions

The method provides a powerful tool for studying live cell interactions in the intestine.
It enhances understanding of the role of immune cells in mucosal biology.
Further applications could extend to cancer biology and other areas.

Frequently Asked Questions

What is intravital microscopy?

Intravital microscopy is a technique that allows for the visualization of live cells within a living organism, providing insights into cellular dynamics.

How long can cells be tracked using this method?

Cells can be tracked for up to 4 hours using this imaging technique.

What is the significance of using GFP-labeled cells?

GFP labeling allows for the visualization of specific cell types, enabling researchers to study their behavior and interactions in real-time.

What challenges are associated with this technique?

Challenges include mastering the surgical technique and ensuring optimal positioning of the mouse for image acquisition.

What insights can be gained from this method?

This method provides insights into mucosal immunity, epithelial biology, and cellular interactions within the intestine.

What type of mice are used in this study?

Transgenic mice expressing enhanced green fluorescent protein (EGFP) are used for visualization.

Nós descrevemos um método para visualizar o IELS do ltγδ GFP-etiquetado usando a imagem latente intravital do intestino pequeno murino pela microscopia confocal invertida do disco de giro. Esta técnica permite o rastreamento de células ao vivo dentro da mucosa para até 4 h e pode ser usada para investigar uma variedade de interações imunológicas intestinais.

A microscopia intravital da pequena mucosa intestinal pode fornecer uma visão da dinâmica espesso das interações celulares dentro do epitélio entre diferentes tipos de células imunes, ou entre esses dois compartimentos. A principal vantagem dessa técnica é que ela permite a aquisição de imagens de alta velocidade e alta resolução dentro da mucosa intestinal em tempo real. Imagens ao vivo do intestino podem fornecer informações adicionais sobre as interações celulares, ou sinalização celular envolvida na imunidade mucosa, biologia epitelial ou biologia do câncer.

Trata-se de um método desafiador que requer a prática da técnica cirúrgica, bem como paciência e posicionamento ideal do mouse para aquisição de imagem. Depois de confirmar a falta de resposta ao beliscão do dedo do dedo em um rato transgênico anestesiado, expressando uma proteína fluorescente verde aprimorada, ou EGFP, receptor de células T Gamma-delta, use uma seringa tuberculina para injetar lentamente 200 microliters de Hoechst 33342 Dye Solution recém-preparada no seio venoso retro-orbital. De um a dois minutos após a injeção, aplique pomada nos olhos do animal para evitar que eles sequem.

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