Universität zu Köln

Herb, Marc ORCID: 0000-0002-7533-0288, Farid, Alina, Gluschko, Alexander, Kroenke, Martin and Schramm, Michael (2019). Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA. J. Vis. Exp. (153). CAMBRIDGE: JOURNAL OF VISUALIZED EXPERIMENTS. ISSN 1940-087X

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Abstract

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Herb, Marc UNSPECIFIED orcid.org/0000-0002-7533-0288 UNSPECIFIED Farid, Alina UNSPECIFIED UNSPECIFIED UNSPECIFIED Gluschko, Alexander UNSPECIFIED UNSPECIFIED UNSPECIFIED Kroenke, Martin UNSPECIFIED UNSPECIFIED UNSPECIFIED Schramm, Michael UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-128937
DOI:	10.3791/60143
Journal or Publication Title:	J. Vis. Exp.
Number:	153
Date:	2019
Publisher:	JOURNAL OF VISUALIZED EXPERIMENTS
Place of Publication:	CAMBRIDGE
ISSN:	1940-087X
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language EXPRESSION; RECEPTORS Multiple languages Multidisciplinary Sciences Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/12893