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

Materials and Reagents

1. 0.1-10 μl pipet tips (Thermo Fisher Scientific, Thermo Scientific^TM, 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, Invitrogen^TM, 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, Invitrogen^TM, 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, Gibco^TM, catalog number: 21051-024 )
    
60. NaHCO₃ (KANTO KAGAKU, catalog number: 37116-00 )
    
61. Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, 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 StepOnePlus^TM Real-Time PCR systems (Thermo Fisher Scientific, Applied Biosystems^TM, 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 C_T 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.