Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

Misty M. Montoya; K. Mark Ansel

doi:10.3791/55546

Kleine RNA Transfektion in primären humanen Th17-Zellen durch Next Generation Elektroporation

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

Kleine RNA Transfektion in primären humanen Th17-Zellen durch Next Generation Elektroporation

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10:15 min

April 13, 2017

DOI: 10.3791/55546-v

Misty M. Montoya¹, K. Mark Ansel¹

¹Department of Microbiology & Immunology, Sandler Asthma Basic Research Center,University of California, San Francisco

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Overview

This article discusses next generation electroporation as an efficient method for transfecting human Th17 cells with small RNAs. This technique aims to enhance understanding of gene expression and T cell behavior.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Immunology

Background

Next generation electroporation is a reliable transfection method.
It targets primary human T cells for gene expression studies.
Understanding T cell differentiation is crucial for immune function research.
This method can be applied to various lymphocyte systems.

Purpose of Study

To efficiently transfect primary human T cells with small RNA reagents.
To investigate the regulation of T cell differentiation.
To explore the immune function of Th17 cells.

Methods Used

Coating tissue culture plates with antihuman CD3 and CD28.
Isolating cord-blood mononuclear cells (CBMCs).
Performing CD4 positive T cell isolation by negative selection.
Utilizing small RNA reagents for transfection.

Main Results

Demonstrated high efficiency in transfecting Th17 cells.
Provided insights into gene regulation in T cells.
Showed applicability to other lymphocyte types.
Enhanced understanding of immune responses.

Conclusions

Next generation electroporation is a valuable tool in T cell research.
This method can significantly advance studies in immunology.
Future applications may extend to various cell types and conditions.

Frequently Asked Questions

What is next generation electroporation?

It is an efficient method for transfecting cells with small RNAs.

How does this method benefit T cell research?

It allows for precise manipulation of gene expression in T cells.

Can this technique be used on other cell types?

Yes, it can be applied to various lymphocytes from humans or mice.

What are the main advantages of this technique?

It is highly efficient and reliable for transfection.

What is the significance of studying Th17 cells?

Th17 cells play a crucial role in immune responses and inflammation.

What reagents are used in this method?

Small RNA reagents and synthetic small RNAs are utilized.

Die Elektroporation der nächsten Generation ist eine effiziente Methode zur Transfektion menschlicher Th17-Zellen mit kleinen RNAs, um die Genexpression und das Zellverhalten zu verändern.

Das übergeordnete Ziel dieses Verfahrens ist die effiziente Transfektion primärer humaner T-Zellen mit kleinen RNA-Reagenzien unter Verwendung der Elektroporation der nächsten Generation. Diese Methode kann helfen, wichtige Fragen der T-Zellbiologie zu beantworten, z. B. wie bestimmte Genprodukte die T-Zell-Differenzierung und die Immunfunktion regulieren. Der Hauptvorteil dieser Technik besteht darin, dass es sich um eine hocheffiziente und zuverlässige Methode zur Transfektion primärer humaner T-Zellen mit sRNAs und anderen synthetischen kleinen RNA-Reagenzien handelt.

Diese Methode kann Einblicke in die Biologie menschlicher Th17-Zellen geben, aber sie kann auch auf andere Systeme angewendet werden, wie z.B. andere Lymphozyten von Menschen oder Mäusen. Beschichten Sie am Tag Null sechs Well-Gewebekulturplatten mit 1,5 Millilitern Antihuman-CD3 und Antihuman-CD28 in PBS mindestens zwei Stunden lang bei 37 Grad Celsius mit Calcium und Magnesium. Nachdem die mononukleären Nabelschnurblutzellen (CBMCs) isoliert und gewaschen wurden, führen Sie die Isolierung von CD4-positiven T-Zellen durch negative Selektion mit einem kommerziellen humanen CD4-positiven T-Zell-Kit durch.

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