采用PHA活化和Neon电穿孔法转染人幼稚CD4+T淋巴细胞

Amy Palin; David B. Lewis

doi:10.21769/BioProtoc.836

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Peer-reviewed

Transfection of Human Naive CD4⁺ T Cells with PHA Activation and Neon Electroporation

采用PHA活化和Neon电穿孔法转染人幼稚CD4+T淋巴细胞

Amy Palin

DL David B. Lewis email

发布: 2013年08月05日第3卷第15期 DOI: 10.21769/BioProtoc.836 浏览次数: 17340

评审: Anonymous reviewer(s)

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Cover of The Journal of Immunology, featuring study using the protocol.

Mar 2013

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Abstract

Transfection of primary T cells can be challenging. This protocol describes a method to transfect primary human naive CD4⁺ T cells with an AP-1 luciferase reporter using low-level activation by phytohemagglutinin (PHA) and electroporation, as published (Palin et al., 2013). This technique is a modification of one previously described by our group (Cron et al., 2013). Anyone wishing to transfect murine T cells should consult the publication by Cron et al., 2013. This technique may be adapted for other primary T cell types by optimizing the Neon electroporation conditions, as described in the text. Other luciferase or GFP reporters may be used, and will require optimization of the stimulation conditions for that particular reporter.

Keywords: Human CD4+ T cells (人体的CD4 + T细胞)

Transfection (转染)

Electroporation (电穿孔)

Luciferase reporter (荧光素酶报告基因)

GFP reporter (GFP报告)

Materials and Reagents

Human blood or peripheral blood mononuclear cells (PBMCs), collected using heparin (1 ml per 60 ml of blood as an anti-coagulant)
Heparin (Sigma-Aldrich, catalog number: H3393 )
Ficoll-Hypaque (GE Life Sciences, catalog number: 17-1440-02 )
Human MACS Naive CD4⁺ T cell II Kit (Miltenyi Biotec, catalog number: 130-094-131 )
BSA (Thermo Fisher Scientific, catalog number: SH30574 )
PBS (Life Technologies, Invitrogen^TM, catalog number: 10010 )
RPMI medium (Life Technologies, Invitrogen^TM, catalog number: 11875093 )
Heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals or other supplier)
Hank's balanced salt solution without calcium or magnesium (HBSS) (Life Technologies, Invitrogen^TM, catalog number: 14170161 )
PHA (Sigma-Aldrich, catalog number: 61764 )
Neon Transfection System 100 μl kit (Life Technologies, Invitrogen^TM, catalog number: MPK10096 or MPK10025 )
Aqua Amine Live/Dead discriminator (Life Technologies, Invitrogen^TM, catalog number: L34957 )
7AAD (BD Biosciences, catalog number: 559925 )
Anti-CD3⁺ anti-CD28^- coated Dynal beads (Life Technologies, Invitrogen^TM, catalog number: 11131D )
Ionomycin (Sigma-Aldrich, catalog number: I3909 )
Phorbol myristate acetate (PMA) (Sigma-Aldrich, catalog number: P8139 )
One Glo luciferase reagent (Promega Corporation, catalog numbers: E6110 , E6120 , or E6130 )
AP-1-luciferase reporter (as described in Vaysberg et al., 2008)
Note: This plasmid consists of a pGL3 backbone (Promega Corporation, catalog number: E1751) with 5 copies of the AP-1 binding site from the metallothionein promoter inserted into the human IL2 minimal promoter.
pEGFP-N1 (Clonetech) or equivalent GFP-expressing plasmid
Beta-actin (b-actin)-driven luciferase reporter plasmid, or other highly expressing luciferase reporter
Note: Use of a renilla luciferase-expressing plasmid is not recommended in this assay, as we found it interferes with the firefly luciferase signal.
CD3-Alexa700 (eBiosceince, catalog number: 56-0037-42 )
CD4-PE-Cy7 (BD Biosciences, catalog number: 557852 )
CD4-PE (Life Technologies, Invitrogen^TM, catalog number: MHCD0404 )
CD45RA–PacificBlue (Life Technologies, Invitrogen^TM, catalog number: MHCD45RA28 )
CD45RO-PerCp-Cy5.5 (BD Biosciences, catalog number: 560607 )
CD4RA-APC (Life Technologies, Invitrogen^TM, catalog number: MHCD45RA05 ) or Alexa647 (BD Biosciences, catalog number: 562763 )
CD25-APC (Life Technologies, Invitrogen^TM, catalog number: MHCD2505 )
CD40L-FITC (BD Biosciences, catalog number: 555699 )
CD62L-PE (BD Biosciences, catalog number: 555544 )
CD69-PE-Cy5 (Life Technologies, Invitrogen^TM, catalog number: MHCD6906 )
MACS buffer (see Recipes)

Equipment

96-well round-bottom sterile tissue culture plates (BD Biosciences, catalog number: 353077 or equivalent)
96-well white-wall, flat-bottom plates (BD Biosciences, catalog number: 353296 or equivalent)
MACS LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
Neon Transfection System (Life Technologies, Invitrogen^TM, model: MPK5000 )
Dynal magnet (Life Technologies, Invitrogen^TM, model: 12321D )
Luminometer with 96-well plate capability (any manufacturer)
MACS midi magnets (Miltenyi Biotec, model: 130-042-302 )
Flow cytometer (BD LSR II or similar)

Procedure

English

中文翻译

文章信息

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如何引用

Palin, A. and Lewis, D. B. (2013). Transfection of Human Naive CD4⁺ T Cells with PHA Activation and Neon Electroporation. Bio-protocol 3(15): e836. DOI: 10.21769/BioProtoc.836.