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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   

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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

  1. 0.1-10 μl pipet tips (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: QSP#TF104 )
  2. 1-200 μl pipet tips (Corning, catalog number: 4845 )
  3. 100-1,000 μl pipet tips (Corning, catalog number: 4846 )
  4. 15 ml centrifuge tubes (Corning, Falcon®, catalog number: 352096 )
  5. 50 ml centrifuge tubes (Corning, Falcon®, catalog number: 352070 )
  6. 50 ml syringe (NIPRO, catalog number: 4987458089534 )
  7. 0.20 μm sterilizing filter (Advantec MFS, catalog number: 25CS020AS )
  8. Falcon 12-well tissue culture plate (Corning, Falcon®, catalog number: 353043 )
  9. Seamless cellulose tubing (EIDIA, catalog number: 410490033 )
  10. 96-well fast plate (NIPPON Genetics, catalog number: 38801 )
  11. qPCR adhesive seal (NIPPON Genetics, catalog number: 4Ti-0560 )
  12. Mouse embryonic fibroblasts (MEFs) of C57/B6 origin (E13.5) (Bryja et al., 2006)
  13. CD14 microbeads, human (Miltenyi Biotec, catalog number: 130-050-201 )
  14. 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
  15. ISOGEN (Nippon Gene, catalog number: 311-02501 )
  16. Nuclease free-H2O (as an accessory reagent of ReverTra Ace qRT-PCR Kit) (TOYOBO, catalog number: FSQ-101 )
  17. DNase I (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18068015 )
  18. EDTA
  19. ReverTra Ace qRT-PCR Kit (TOYOBO, catalog number: FSQ-101)
  20. SYBR Premix Ex Taq (2x) (Tli RNase H Plus) (Takara Bio, catalog number: RR420 )
  21. ROX reference dye (50x) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 12223-012 )
  22. Primers for amplification of mouse TIPARP cDNA by quantitative PCR (Sigma-Aldrich):
    Forward: 5’-GCCAGACTGTGTAGTACAGCC-3’
    Reverse: 5’-GGGTTCCAGTTCCCAATCTTTT-3’
  23. Primers for amplification of mouse ACTB cDNA by quantitative PCR (Sigma-Aldrich):
    Forward: 5’-AGTGTGACGTTGACATCCGTA-3’
    Reverse: 5’-GCCAGAGCAGTAATCTCCTTCT-3’
  24. Primers for amplification of human TIPARP cDNA by quantitative PCR (Sigma-Aldrich):
    Forward: 5’-GTTGGGGACCAGATACCGGA-3’
    Reverse: 5’-CTGGGTGCAAAAGATCAGTCT-3’
  25. Primers for amplification of human GAPDH cDNA by quantitative PCR (Sigma-Aldrich):
    Forward: 5’-CATGAGAAGTATGACAACAGCCT-3’
    Reverse: 5’-AGTCCTTCCACGATACCAAAGT-3’
  26. NaCl
  27. NaH2PO4·H2O
  28. KCl
  29. CaCl2
  30. MgSO4·7H2O
  31. Fe(NO3)3·9H2O
  32. L-arginine·HCl
  33. L-histidine·HCl·H2O
  34. L-isoleucine
  35. L-leucine
  36. L-lysine·HCl
  37. L-methionine
  38. L-phenylalanine
  39. L-threonine
  40. Glycine
  41. L-valine
  42. L-cysteine·HCl·H2O
  43. L-serine
  44. L-tyrosine
  45. Choline bitartrate
  46. Folic acid
  47. D-Ca pantothenate
  48. Myo-Inositol
  49. Niacinamide (Nicotinamide)
  50. Pyridoxal·HCl
  51. Thiamine·HCl
  52. Riboflavin
  53. D-glucose
  54. Sodium pyruvate
  55. Phenol Red Na
  56. Succinic acid
  57. Disodium succinate
  58. Dulbecco’s modified Eagle’s medium (DMEM) (NISSUI PHARMACEUTICAL, catalog number: 05919 )
  59. L-glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 21051-024 )
  60. NaHCO3 (KANTO KAGAKU, catalog number: 37116-00 )
  61. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437-028 )
  62. 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 )
  63. Phosphate-buffered saline (PBS) (pH 7.4) (NISSUI PHARMACEUTICAL, catalog number: 05913 )
  64. Dimethylsulfoxide (DMSO) (Dojindo Molecular Technologies, catalog number: SP10 )
  65. Isopropanol (NACALAI TESQUE, catalog number: 29113-53 )
  66. Ethanol (99.5%) (NACALAI TESQUE, catalog number: 14713-95 )
  67. Chloroform (KANTO CHEMICAL, catalog number: 07278-00 )
  68. Tryptophan-free (Trp-free) DMEM (Cell Science & Technology Institute, special order) (see Recipes)
  69. DMEM (FBS+) (see Recipes)
  70. Trp-free DMEM (FBS+) (see Recipes)
  71. Kyn stock solution
  72. FICZ stock solution

Equipment

  1. Pipettes (PIPETMAN P2) (Gilson, catalog number: F144801 )
  2. Pipettes (PIPETMAN P20) (Gilson, catalog number: F123600 )
  3. Pipettes (PIPETMAN P1000) (Gilson, catalog number: F123602 )
  4. Bio clean bench (Hitachi, model: 1305BNG3-AG )
  5. Labnet VX100 vortex (Labnet International, catalog number: 13111-LV2 )
  6. 37 °C and 5% CO2 cell culture incubator (WAKENBTECH, catalog number: 9000EX )
  7. ABI StepOnePlusTM Real-Time PCR systems (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4379216 )

Procedure

  1. Treatment with Kyn or FICZ (each sample is usually prepared in triplicate to assess reproducibility)
    1. Seed MEFs or primary human monocytes to 12-well plate as 1 x 106 cells/well with 500 μl of DMEM (FBS+) and culture them at 37 °C in a 5% CO2-incubator.
    2. After 24 h, MEFs are washed once with 1 ml of pre-warmed PBS, and incubated with 500 μl of Trp-free DMEM (FBS+) for 24 h at 37 °C. This step is performed to cultivate cells under Trp-free conditions. This Trp-free culturing does not affect the growth of MEFs at least under these conditions.
    3. Remove medium, and incubate cells with 500 μl of Trp-free DMEM (FBS+) containing Kyn (0, 50, 100 or 200 μM) or FICZ (0, 0.1, 1 or 25 nM) for 2 h at 37 °C.
  2. To evaluate the effect of AHR ligands, the mRNA induction of TIPARP, one of the AHR-inducible genes, is analyzed by qRT-PCR as below:
    1. At 2 h after treatment of AHR ligands, cells are washed with PBS and dissolved by directly adding 500 μl of ISOGEN. Total RNA is then extracted according to the manufacturer’s protocol and adjusted to 1 μg/μl with nuclease free-H2O.
    2. In this step, total RNA is purified by degrading contaminated DNA with DNase I treatment. Make a mixture in the following order:


    3. Incubate the mixture samples at 37 °C for 15 min.
    4. Add 1 μl EDTA to each sample and incubate the samples at 65 °C for 10 min. This step is needed to stop the activity of DNase I.
    5. To next synthesize cDNA with a ReverTra Ace qRT-PCR Kit, make a mixture with each premixed reagent that is included in the kit, in the following order:


    6. Incubate the mixture samples at 37 °C for 15 min, and then incubate them at 98 °C for 5 min.
    7. Make a 4-fold dilution of the cDNA samples with nuclease free-H2O for each qPCR template.
    8. Expression levels of reverse-transcribed mRNA, that is, cDNA, in each sample are next evaluated by qPCR analysis, wherein SYBR, a DNA-binding fluorescent dye, is used for the measurement of the levels of amplified DNAs, while ROX is used as a reference dye. Set up the following reaction mixture to amplify cDNA:

      Note: Use 1 μl of nuclease free-H2O or no-RT samples instead of a DNA sample as a negative control to confirm the procedure works as expected.
    9. The PCR plate is covered with an adhesive transparent cover, and then centrifuged shortly.
    10. Set the plate in the real-time instrument and start the real-time PCR following the program as below:

Data analysis

  1. TIPARP mRNA levels are normalized to the mRNA expression levels of ACTB or GAPDH for each sample by ∆∆Ct methods (Schmittgen and Livak, 2008).
  2. Statistical significance between two samples is determined by Student’s t-test.

Recipes

  1. Tryptophan-free DMEM

    Table 1. Composition of Trp-free DMEM. This is prepared by special order (Cell Science & Technology Institute).


  2. DMEM (FBS+)
    DMEM is supplemented with 4 mM L-glutamine, 0.1% NaHCO3 and 10% heat inactivated FBS
  3. Trp-free DMEM (FBS+)
    1. To deplete tryptophan from FBS, FBS is dialyzed against 100-fold volumes of PBS at 4 °C for 24 h using seamless cellulose tubing with exchange of the dialysis solution
    2. Trp-free DMEM is supplemented with 4 mM L-glutamine, 0.1% NaHCO3 and 10% heat inactivated and dialyzed FBS
  4. Kyn stock solution
    Kyn is dissolved at 25 mM in PBS
  5. FICZ stock solution
    FICZ is dissolved at 10 mg/ml (35.2 mM) in DMSO 

Acknowledgments

This protocol, which was used in Figure 3b and Supplementary Figure 2g of Yamada et al. (2016), is based on the earlier work by Opitz et al. (2011). This was supported by a grant from the Ministry of Health, Labour and Welfare of Japan, the Ministry of Education, Culture, Sports, Science and Technology of Japan (grant-in-aid for scientific research (A) [25253030] and grant-in-aid for scientific research on innovative areas [25115502, 23112701]), the Kato Memorial Bioscience Foundation, the Yasuda Medical Foundation, the Takeda Science Foundation and the Waksman Foundation of Japan to A.T.

References

  1. Bryja, V., Bonilla, S. and Arenas, E. (2006). Derivation of mouse embryonic stem cells. Nat Protoc 1(4): 2082-2087.
  2. Ma, Q. (2002). Induction and superinduction of 2,3,7,8-tetrachlorodibenzo-rho-dioxin-inducible poly(ADP-ribose) polymerase: role of the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator transcription activation domains and a labile transcription repressor. Arch Biochem Biophys 402(2): 309-316.
  3. Opitz, C. A., Litzenburger, U. M., Sahm, F., Ott, M., Tritschler, I., Trump, S., Schumacher, T., Jestaedt, L., Schrenk, D., Weller, M., Jugold, M., Guillemin, G. J., Miller, C. L., Lutz, C., Radlwimmer, B., Lehmann, I., von Deimling, A., Wick, W. and Platten, M. (2011). An endogenous tumour-promoqting ligand of the human aryl hydrocarbon receptor. Nature 478(7368): 197-203.
  4. Schmittgen, T. D. and Livak, K. J. (2008). Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3(6): 1101-1108.
  5. Smirnova, A., Wincent, E., Vikström Bergander, L., Alsberg, T., Bergman, J., Rannug, A. and Rannug, U. (2016). Evidence for new light-independent pathways for generation of the endogenous aryl hydrocarbon receptor agonist FICZ. Chem Res Tpxicol 29: 75-86.
  6. Stockinger, B., Di Meglio, P., Gialitakis, M. and Duarte, J. H. (2014). The aryl hydrocarbon receptor: multitasking in the immune system. Annu Rev Immunol 32: 403-432.
  7. Yamada, T., Horimoto, H., Kameyama, T., Hayakawa, S., Yamato, H., Dazai, M., Takada, A., Kida, H., Bott, D., Zhou, A. C., Hutin, D., Watts, T. H., Asaka, M., Matthews, J. and Takaoka, A. (2016). Constitutive aryl hydrocarbon receptor signaling constrains type I interferon-mediated antiviral innate defense. Nat Immunol 17(6): 687-694.

简介

通过内源性配体活化芳基烃受体(AHR)已涉及多种生理过程,如细胞周期调控,细胞分化和免疫应答。据报道,色氨酸代谢物,如犬尿胆碱(Kyn)和6-甲基吲哚(3,2-b)咔唑(FICZ)是AHR的内源性配体(Stockinger等人,2014)。该方案设计用于在小鼠胚胎成纤维细胞(MEF)或初级周边单核细胞中用Kyn或FICZ进行治疗。

背景 色氨酸代谢物如Kyn和FICZ是生理条件下AHR的内源性配体。 Kyn由色氨酸-2,3-双加氧酶(TDO)和/或吲哚胺-2,3-双加氧酶1和2(IDO1 / 2)产生,并有助于抑制抗肿瘤反应和恶性进展(Stockinger等人,2014)。 FICZ通过将L-色氨酸暴露于紫外线B照射而产生,并且涉及许多生物过程(Smirnova等人,2016)。在适应性免疫系统中,FICZ显示出促进Th17细胞反应(Stockinger等人,2014)。还已经显示通过用这些内源性AHR配体进行治疗来抑制病毒感染期间的先天性干扰素反应(Yamada等人,2016)。为了评估通过用这些配体处理的AHR活化的影响,使用无色氨酸培养基和透析的FBS在不含色氨酸的条件下培养细胞(Opitz等人,2011)。分析TCDD诱导型多聚(ADP-核糖)聚合酶(TIPARP)(Ma,2002),一种AHR诱导型基因的检测,以验证配体诱导的AHR活化。

关键字:芳烃受体, 犬尿氨酸, 6-甲酰基吲哚并(3,2-b)咔唑, 色氨酸, TCDD诱导型聚(ADP-核糖)聚合酶

材料和试剂

  1. 0.1-10μl移液管吸头(Thermo Fisher Scientific,Thermo Scientific TM,目录号:QSP#TF104)
  2. 1-200μl移液管提示(康宁,目录号:4845)
  3. 100-1,000μl移液管吸头(康宁,目录号:4846)
  4. 15ml离心管(Corning,Falcon ®,目录号:352096)
  5. 50ml离心管(Corning,Falcon ®,目录号:352070)
  6. 50ml注射器(NIPRO,目录号:4987458089534)
  7. 0.20μm消毒过滤器(Advantec MFS,目录号:25CS020AS)
  8. Falcon 12孔组织培养板(Corning,Falcon ®,目录号:353043)
  9. 无缝纤维素管(EIDIA,目录号:410490033)
  10. 96孔快板(NIPPON Genetics,目录号:38801)
  11. qPCR粘合密封(NIPPON Genetics,目录号:4Ti-0560)
  12. C57/B6起源的小鼠胚胎成纤维细胞(MEFs)(E13.5)(Bryja等人,2006)
  13. CD14微珠,人(Miltenyi Biotec,目录号:130-050-201)
  14. 根据制造商的说明书,通过用CD14微珠进行磁激活细胞分选,从健康志愿者的外周血中分离初级人类外周单核细胞。
  15. ISOGEN(Nippon Gene,目录号:311-02501)
  16. 核酸酶游离H 2 O(作为ReverTra Ace qRT-PCR试剂盒的辅助试剂)(TOYOBO,目录号:FSQ-101)
  17. DNase I(Thermo Fisher Scientific,Invitrogen TM,目录号:18068015)
  18. EDTA
  19. ReverTra Ace qRT-PCR试剂盒(TOYOBO,目录号:FSQ-101)
  20. SYBR Premix Ex Taq(2x)(Tli RNase H Plus)(TaKara Bio,目录号:RR420)
  21. ROX参考染料(50x)(Thermo Fisher Scientific,Invitrogen TM,目录号:12223-012)
  22. 通过定量PCR扩增小鼠TIPARP cDNA的引物(Sigma-Aldrich):
    正向:5'-GCCAGACTGTGTAGTACAGCC-3'
    反向:5'-GGGTTCCAGTTCCCAATCTTTT-3'
  23. 通过定量PCR扩增小鼠ACTB cDNA的引物(Sigma-Aldrich):
    正向:5'-AGTGTGACGTTGACATCCGTA-3'
    反向:5'-GCCAGAGCAGTAATCTCCTTCT-3'
  24. 通过定量PCR扩增人TIPARP cDNA的引物(Sigma-Aldrich):
    前进:5'-GTTGGGGACCAGATACCGGA-3'
    反向:5'-CTGGGTGCAAAAGATCAGTCT-3'
  25. 通过定量PCR扩增人GAPDH cDNA的引物(Sigma-Aldrich):
    转发:5'-CATGAGAAGTATGACAACAGCCT-3'
    反向:5'-AGTCCTTCCACGATACCAAAGT-3'
  26. NaCl
  27. NaH 2 PO 4 H 2 O 2/
  28. KCl
  29. CaCl 2
  30. MgSO 4·7H 2 O→//
  31. Fe(NO 3 3)3 2 O
  32. L-精氨酸·HCl
  33. L-组氨酸·HCl·H 2 O
  34. L-异亮氨酸
  35. L-亮氨酸
  36. L-赖氨酸·HCl
  37. L-甲硫氨酸
  38. L-苯丙氨酸
  39. L-苏氨酸
  40. 大豆
  41. L-缬氨酸
  42. L-半胱氨酸·HCl·H 2 O
  43. L-丝氨酸
  44. L-酪氨酸
  45. 酒石酸胆碱
  46. 叶酸
  47. D-Ca泛酸盐
  48. 肌醇
  49. 烟酰胺(烟酰胺)
  50. 吡哆醛·HCl
  51. 硫胺素·HCl
  52. 核黄素
  53. D-葡萄糖
  54. 丙酮酸钠
  55. 酚红钠
  56. 琥珀酸
  57. 琥珀酸二钠
  58. Dulbecco改良Eagle's培养基(DMEM)(NISSUI PHARMACEUTICAL,目录号:05919)
  59. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:21051-024)
  60. NaHCO 3(KANTO KAGAKU,目录号:37116-00)
  61. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10437-028)
  62. (Kyn)(Sigma-Aldrich,目录号:K8625)和6-甲基吲哚(3,2-b)咔唑(FICZ)(Enzo Life Sciences,目录号:BML-GR206-0100)的储备溶液/>
  63. 磷酸盐缓冲盐水(PBS)(pH 7.4)(NISSUI PHARMACEUTICAL,目录号:05913)
  64. 二甲基亚砜(DMSO)(Dojindo Molecular Technologies,目录号:SP10)
  65. 异丙醇(NACALAI TESQUE,目录号:29113-53)
  66. 乙醇(99.5%)(NACALAI TESQUE,目录号:14713-95)
  67. 氯仿(KANTO CHEMICAL,目录号:07278-00)
  68. 不含色氨酸(Trp-free)DMEM(细胞科学与技术研究所,特殊订单)(参见食谱)
  69. DMEM(FBS +)(见食谱)
  70. 无Trp的DMEM(FBS +)(参见食谱)
  71. Kyn股票解决方案
  72. FICZ库存解决方案

设备

  1. 移液器(PIPETMAN P2)(Gilson,目录号:F144801)
  2. 移液器(PIPETMAN P20)(Gilson,目录号:F123600)
  3. 移液器(PIPETMAN P1000)(Gilson,目录号:F123602)
  4. 生物清洁工作台(日立,型号:1305BNG3-AG)
  5. Labnet VX100涡流(Labnet International,目录号:13111-LV2)
  6. 37℃和5%CO 2细胞培养箱(WAKENBTECH,目录号:9000EX)
  7. ABI StepOnePlus TM实时PCR系统(Thermo Fisher Scientific,Applied Biosystems TM,目录号:4379216)

程序

  1. 用Kyn或FICZ处理(每个样品通常准备一式三份以评估重现性)
    1. 将种子MEF或原代人单核细胞以1×10 6个细胞/孔加入到12孔板中,加入500μlDMEM(FBS +),并在37℃,5%CO 2 -incubator。
    2. 24小时后,用1ml预热的PBS洗涤MEF一次,并在37℃下与500μl无Trp的DMEM(FBS +)孵育24小时。执行该步骤以在无Trp条件下培养细胞。至少在这些条件下,这种无Trp培养不影响MEFs的生长
    3. 取出培养基,并在37℃下将含有Kyn(0,50,100或200μM)或FICZ(0,0.1,1或25nM)的500μl含Trp的DMEM(FBS +)孵育细胞2小时。 br />
  2. 为了评估AHR配体的作用,通过qRT-PCR分析了其中一个AHR诱导型基因TIPARP的mRNA诱导,如下所示:
    1. 处理AHR配体2小时后,用PBS洗涤细胞,直接加入500μl的ISOGEN溶解。然后根据制造商的方案提取总RNA,并用核酸酶自由-H 2 O调节至1μg/μl。
    2. 在该步骤中,通过用DNA酶I处理降解污染的DNA来纯化总RNA。按以下顺序混合:


    3. 将混合物样品在37℃下孵育15分钟
    4. 向每个样品加入1μlEDTA,并在65°C下孵育样品10分钟。需要这一步来停止DNase I的活动。
    5. 接下来用ReverTra Ace qRT-PCR试剂盒合成cDNA,按照以下顺序与试剂盒中包含的每种预混试剂混合:


    6. 将混合物样品在37℃孵育15分钟,然后在98℃下孵育5分钟。
    7. 对于每个qPCR模板,使用无核酸酶free-H 2 O的cDNA样品进行4倍稀释。
    8. 接下来通过qPCR分析评估每个样品中逆转录mRNA,即cDNA的表达水平,其中使用SYBR,DNA结合荧光染料测量扩增DNA的水平,而ROX用作参考染料。设置以下反应混合物以扩增cDNA:

      注意:使用1μl核酸酶游离H 2 O或无RT样品代替DNA样品作为阴性对照,以确认程序按预期进行。
    9. PCR板用粘合剂透明盖子覆盖,然后立即离心。
    10. 在实时仪器中设置板块,并按照以下程序开始实时PCR:

数据分析

  1. 通过ΔΔCt方法,将TIPARP mRNA水平标准化为每个样品的ACTB或GAPDH的mRNA表达水平(Schmittgen和Livak,2008)。
  2. 两个样本之间的统计学意义由Student's 测试确定。

食谱

  1. 不含色氨酸的DMEM

    表1.无Trp的DMEM的组成这是由特殊订单(Cell Science& Technology Institute)制备的。


  2. DMEM(FBS +)
    补充有DMEM的4mM L-谷氨酰胺,0.1%NaHCO 3和10%热灭活的FBS
  3. 无Trp的DMEM(FBS +)
    1. 为了从FBS中消耗色氨酸,FBS在4℃下用100倍体积的PBS透析24小时,使用无缝纤维素管与交换透析溶液
    2. 无Trp的DMEM补充有4mM L-谷氨酰胺,0.1%NaHCO 3和10%热灭活和透析的FBS
  4. Kyn股票解决方案
    Kyn在PBS中以25mM溶解
  5. FICZ库存解决方案
    FICZ以DMSO/
    中的10mg/ml(35.2mM)溶解

致谢

该协议在Yamada等人的图3b和补充图2g中使用。 (2016)基于Opitz等人早期的研究工作。 (2011)。这得到了日本卫生部,劳动福利部,日本教育部,文化部,体育部,科技部(赠予援助科学研究(A))[25253030]和赠款的资助,对创新领域的科学研究援助[25115502,23112701]),加藤纪念生物科学基金会,安田医学基金会,武田科学基金会和日本瓦克斯曼基金会对AT

参考文献

  1. Bryja,V.,Bonilla,S.和Arenas,E.(2006)。小鼠胚胎干细胞的衍生 Nat Protoc 1(4):2082-2087。
  2. Ma,Q.(2002)。  诱导和超导2,3,7,8-四氯二苯并呋喃二恶英诱导型聚(ADP-核糖)聚合酶:芳基烃受体/芳烃受体核转运转录激活结构域的作用和不稳定的转录抑制子。 > Arch Biochem Biophys 402(2):309-316。
  3. Opitz,CA,Litzenburger,UM,Sahm,F.,Ott,M.,Tritschler,I.,Trump,S.,Schumacher,T.,Jestaedt,L.,Schrenk,D.,Weller,M.,Jugold, M.,Guillemin,GJ,Miller,CL,Lutz,C.,Radlwimmer,B.,Lehmann,I.,von Deimling,A.,Wick,W。和Platten,M。(2011)。 ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/21976023"target ="_ blank">人类芳烃受体的内源性肿瘤促进配体。自然 478(7368):197-203。
  4. Schmittgen,TD和Livak,KJ(2008)。分析实时PCR数据通过比较C< T>方法。 Nat Protoc 3(6):1101-1108。
  5. Smirnova,A.,Wincent,E.,VikströmBergander,L.,Alsberg,T.,Bergman,J.,Rannug,A.and Rannug,U.(2016)。< a class ="ke-insertfile" href ="http://www.ncbi.nlm.nih.gov/pubmed/26686552"target ="_ blank">用于产生内源性芳烃受体激动剂FICZ的新的光依赖途径的证据。 Res Tpxicol 29:75-86。
  6. Stockinger,B.,Di Meglio,P.,Gialitakis,M.and Duarte,JH(2014)。  芳基烃受体:免疫系统中的多任务。 Annu Rev Immunol 32:403-432。
  7. Yamada,T.,Horimoto,H.,Kameyama,T.,Hayakawa,S.,Yamato,H.,Dazai,M.,Takada,A.,Kida,H.,Bott,D.,Zhou,AC,Hutin ,D.,Watts,TH,Asaka,M.,Matthews,J.andTakaoka,A。(2016)。 
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: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.
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