使用内源性配体体外处理小鼠和人类细胞激活芳烃受体

Taisho  Yamada; Akinori Takaoka

doi:10.21769/BioProtoc.2097

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Peer-reviewed

In vitro Treatment of Mouse and Human Cells with Endogenous Ligands for Activation of the Aryl Hydrocarbon Receptor

使用内源性配体体外处理小鼠和人类细胞激活芳烃受体

Taisho Yamada

Akinori Takaoka email

发布: 2017年01月05日第7卷第1期 DOI: 10.21769/BioProtoc.2097 浏览次数: 8118

评审: Ivan ZanoniMeenal SinhaBenoit Chassaing

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Abstract

Activation of the aryl hydrocarbon receptor (AHR) by endogenous ligands has been implicated in a variety of physiological processes such as cell cycle regulation, cell differentiation and immune responses. It is reported that tryptophan metabolites, such as kynurenine (Kyn) and 6-formylindolo(3,2-b)carbazole (FICZ), are endogenous ligands for AHR (Stockinger et al., 2014). This protocol is designed for treatment with Kyn or FICZ in mouse embryonic fibroblasts (MEFs) or primary peripheral monocytes.

Keywords: Aryl hydrocarbon receptor (芳烃受体)

Kynurenine (犬尿氨酸)

6-formylindolo(3,2-b)carbazole (6-甲酰基吲哚并（3,2-b）咔唑)

Tryptophan (色氨酸)

TCDD-inducible poly(ADP-ribose)polymerase (TCDD诱导型聚（ADP-核糖）聚合酶)

Background

Tryptophan metabolites such as Kyn and FICZ are endogenous ligands for AHR under physiological conditions. Kyn is generated by tryptophan-2,3-dioxygenase (TDO) and/or indoleamine-2,3-dioxygenases 1 and 2 (IDO1/2) and contributes to the suppression of antitumor response and malignant progression (Stockinger et al., 2014). FICZ is produced by exposure of L-tryptophan to ultraviolet B irradiation and is involved in many biological processes (Smirnova et al., 2016). In the adaptive immune system, FICZ is shown to promote Th17 cell response (Stockinger et al., 2014). It has also been shown that innate interferon response during viral infection is suppressed by treatment with these endogenous AHR ligands (Yamada et al., 2016). In order to evaluate the effect of AHR activation by treatment with these ligands, tryptophan-free culture medium and dialyzed FBS are used to cultivate cells under tryptophan-free conditions (Opitz et al., 2011). The detection of TCDD-inducible poly(ADP-ribose)polymerase (TIPARP) (Ma, 2002), one of the AHR-inducible genes, is analyzed to verify ligand-induced AHR activation.

Materials and Reagents

0.1-10 μl pipet tips (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: QSP#TF104 )
1-200 μl pipet tips (Corning, catalog number: 4845 )
100-1,000 μl pipet tips (Corning, catalog number: 4846 )
15 ml centrifuge tubes (Corning, Falcon^®, catalog number: 352096 )
50 ml centrifuge tubes (Corning, Falcon^®, catalog number: 352070 )
50 ml syringe (NIPRO, catalog number: 4987458089534 )
0.20 μm sterilizing filter (Advantec MFS, catalog number: 25CS020AS )
Falcon 12-well tissue culture plate (Corning, Falcon^®, catalog number: 353043 )
Seamless cellulose tubing (EIDIA, catalog number: 410490033 )
96-well fast plate (NIPPON Genetics, catalog number: 38801 )
qPCR adhesive seal (NIPPON Genetics, catalog number: 4Ti-0560 )
Mouse embryonic fibroblasts (MEFs) of C57/B6 origin (E13.5) (Bryja et al., 2006)
CD14 microbeads, human (Miltenyi Biotec, catalog number: 130-050-201 )
Primary human peripheral monocytes, which are Isolated from peripheral blood of healthy volunteers by magnetic-activated cell sorting with CD14 microbeads according to the manufacturer’s instructions
ISOGEN (Nippon Gene, catalog number: 311-02501 )
Nuclease free-H₂O (as an accessory reagent of ReverTra Ace qRT-PCR Kit) (TOYOBO, catalog number: FSQ-101 )
DNase I (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 18068015 )
EDTA
ReverTra Ace qRT-PCR Kit (TOYOBO, catalog number: FSQ-101)
SYBR Premix Ex Taq (2x) (Tli RNase H Plus) (Takara Bio, catalog number: RR420 )
ROX reference dye (50x) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 12223-012 )
Primers for amplification of mouse TIPARP cDNA by quantitative PCR (Sigma-Aldrich):
Forward: 5’-GCCAGACTGTGTAGTACAGCC-3’
Reverse: 5’-GGGTTCCAGTTCCCAATCTTTT-3’
Primers for amplification of mouse ACTB cDNA by quantitative PCR (Sigma-Aldrich):
Forward: 5’-AGTGTGACGTTGACATCCGTA-3’
Reverse: 5’-GCCAGAGCAGTAATCTCCTTCT-3’
Primers for amplification of human TIPARP cDNA by quantitative PCR (Sigma-Aldrich):
Forward: 5’-GTTGGGGACCAGATACCGGA-3’
Reverse: 5’-CTGGGTGCAAAAGATCAGTCT-3’
Primers for amplification of human GAPDH cDNA by quantitative PCR (Sigma-Aldrich):
Forward: 5’-CATGAGAAGTATGACAACAGCCT-3’
Reverse: 5’-AGTCCTTCCACGATACCAAAGT-3’
NaCl
NaH₂PO₄·H₂O
KCl
CaCl₂
MgSO₄·7H₂O
Fe(NO₃)₃·9H₂O
L-arginine·HCl
L-histidine·HCl·H₂O
L-isoleucine
L-leucine
L-lysine·HCl
L-methionine
L-phenylalanine
L-threonine
Glycine
L-valine
L-cysteine·HCl·H₂O
L-serine
L-tyrosine
Choline bitartrate
Folic acid
D-Ca pantothenate
Myo-Inositol
Niacinamide (Nicotinamide)
Pyridoxal·HCl
Thiamine·HCl
Riboflavin
D-glucose
Sodium pyruvate
Phenol Red Na
Succinic acid
Disodium succinate
Dulbecco’s modified Eagle’s medium (DMEM) (NISSUI PHARMACEUTICAL, catalog number: 05919 )
L-glutamine (Thermo Fisher Scientific, Gibco^TM, catalog number: 21051-024 )
NaHCO₃ (KANTO KAGAKU, catalog number: 37116-00 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10437-028 )
Stock solutions of L-kynurenine (Kyn) (Sigma-Aldrich, catalog number: K8625 ) and 6-formylindolo(3,2-b)carbazole (FICZ) (Enzo Life Sciences, catalog number: BML-GR206-0100 )
Phosphate-buffered saline (PBS) (pH 7.4) (NISSUI PHARMACEUTICAL, catalog number: 05913 )
Dimethylsulfoxide (DMSO) (Dojindo Molecular Technologies, catalog number: SP10 )
Isopropanol (NACALAI TESQUE, catalog number: 29113-53 )
Ethanol (99.5%) (NACALAI TESQUE, catalog number: 14713-95 )
Chloroform (KANTO CHEMICAL, catalog number: 07278-00 )
Tryptophan-free (Trp-free) DMEM (Cell Science & Technology Institute, special order) (see Recipes)
DMEM (FBS+) (see Recipes)
Trp-free DMEM (FBS+) (see Recipes)
Kyn stock solution
FICZ stock solution

Equipment

Pipettes (PIPETMAN P2) (Gilson, catalog number: F144801 )
Pipettes (PIPETMAN P20) (Gilson, catalog number: F123600 )
Pipettes (PIPETMAN P1000) (Gilson, catalog number: F123602 )
Bio clean bench (Hitachi, model: 1305BNG3-AG )
Labnet VX100 vortex (Labnet International, catalog number: 13111-LV2 )
37 °C and 5% CO₂ cell culture incubator (WAKENBTECH, catalog number: 9000EX )
ABI StepOnePlus^TM Real-Time PCR systems (Thermo Fisher Scientific, Applied Biosystems^TM, catalog number: 4379216 )

Procedure

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Yamada, T. and Takaoka, A. (2017). In vitro Treatment of Mouse and Human Cells with Endogenous Ligands for Activation of the Aryl Hydrocarbon Receptor. Bio-protocol 7(1): e2097. DOI: 10.21769/BioProtoc.2097.