TH17 cells contribute to uveitis and scleritis and are inhibited by IL-27/STAT1 in the retina. (5) Chromatin immunoprecipitation

doi:10.1038/nprot.2007.384

Method Article

TH17 cells contribute to uveitis and scleritis and are inhibited by IL-27/STAT1 in the retina. (5) Chromatin immunoprecipitation

https://doi.org/10.1038/nprot.2007.384

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Biological techniques

Biotechnology

Biochemistry

Computational biology and bioinformatics

experimental autoimmune uveoretinitis (EAU)

IL-27p28 immunohistochemistry and confocal microscopy

intracellular cytokine staining assay

retinal organ culture

*Cheng-Rong Yu prepared this manuscript and should have been the first author.

This protocol is for Chromatin Immunoprecipitation.

Other protocols related to our Nature Medicine paper can be found here:

"Detection of TH17 cells in human blood":http://www.natureprotocols.com/2007/08/30/th17_cells_contribute_to_uveit.php

"Western Blot Analysis":http://www.natureprotocols.com/2007/08/30/th17_cells_contribute_to_uveit_2.php

"Analysis by confocal microscopy":http://www.natureprotocols.com/2007/08/31/th17_cells_contribute_to_uveit_3.php

"Retinal cell isolation":http://www.natureprotocols.com/2007/08/31/th17_cells_contribute_to_uveit_5.php

EZ ChipTM chromatin immunoprecipitation kit (Upstate Biotechnology, Charlottesville, VA)

• Human retinal pigment epithelium cell line (ARPE-19)

• 37% Formaldehyde

• Molecular Biology grade Proteinase K

• Glycogen or tRNA

• 50% Phenol/50%Chloroform (containing 1% isoamyl alcohol)

• Protein G agarose beads (Upstate Biotechnology)

• Protein A Agarose/Salmon sperm DNA (Upstate, Catalog #16-157C)

• Mouse Stat1-specific antibody (Santa Cruz Biotechnology, Santa Cruz, CA)

• IL-27 promoter site-specific primers.

• Solutions

ChIP Dilution Buffer: 0.01% SDS, 1.1% Triton X- 100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl (catalog # 20-153).

Elution buffer: 1% SDS, 0.1 M NaHCO3

Low Salt Immune Complex Wash Buffer: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl. (catalog # 20-154).

High Salt Immune Complex Wash Buffer: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl (catalog # 20-155)

LiCl Immune Complex Wash Buffer: 0.25 M LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA, 10 mM Tris, pH 8.1 (catalog # 20-156).

SDS Lysis Buffer: 10 mL 1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1 (catalog # 20-163).

• Sonicator

1| Crosslink protein/DNA by adding 37% (54 µl) formaldehyde to 2ml of media

2| Shake gently to mix and then incubate at room temperature for 10 min.

3| Add 10X Glycine solution (200 µl) to quench unreacted formaldehyde

4| Mix gently and incubate at room temperature for 5 min.

5| Remove medium and wash with cold PBS (2X)

6| Add 0.1ml PBS containing protease inhibitor cocktail

7| Collect cells, centrifuge at 700xg for 3 min at 4 ^oC and discard supernatant

8| Resuspend cell pellet in 200μl lysis buffer containing protease inhibitor cocktail.

9| Sonicate cell lysate on ice for 4 second 3 times

10| Centrifuge at 14,000 rpm at 4 ⁰C for 10 min and transfer supernatant (~100 μl) to fresh tube

11| Add 900 μl of dilution buffer containing protease inhibitor cocktail

12| Add 60 μl of protein G agarose and incubate for 1 hr at 4 0C with rotation

13| Centrifuge at 3000g for 1 min and collect supernatant into fresh tube

14| Add 4μg of anti STAT1 antibody and incubate overnight at 4 0C with rotation

15| Add 60 μl of protein G agarose for 1 hr at 4 ⁰C with rotation

16| Centrifuge at 3000X g for 1 min and remove supernatant

17| Wash pellet with low salt wash buffer, high salt wash buffer, LiCl immune complex wash buffer and finally TE buffer

18| Add 100 μl of elution buffer and mix gently

19| Incubate at room temperature for 15 min

20| Centrifuge at 3000 x g for 1 min and collect supernatant into new tube

21| Repeat step 19 to 21 and combine supernatant

22| Add 8ul 5M NaCl and incubate at 65 ^oC for 5 hrs

23| Add 1ul of RNase A and incubate for 30 min at 37 ^oC

24| Add 4ul 0.5M EDTA, 8ul 1M Tris-HCl and 1ul Proteinase K and incubate at 45oC for 2 hrs

25| Add 1ml of bind reagent A to each tube and mix well

26| Transfer 600 μl of mixture to spin filter in collection tube and centrifuge at 14,000 g for 30 second

27| Discard liquid in collection tube and put the spin filter back into same collection tube

28| Transfer the remaining 600 μl of mixture to spin filter and centrifuge at 14,000 g for 30 second

29| Remove the liquid in collection tube and add 500 μl of wash reagent B to the spin filter in collection tube

30| Put the spin filter into new collection tube and add 50 μl of elution buffer to the spin filter

31| Centrifuge at 14,000 x g for 30 second and save eluate for PCR

Nussenblatt, R.B. Proctor Lecture. Experimental autoimmune uveitis: mechanisms of disease and clinical therapeutic indications. Invest Ophthalmol Vis Sci 32, 3131-3141 (1991).
Gregerson, D.S., Obritsch, W.F., Fling, S.P. & Cameron, J.D. S-antigen-specific rat T cell lines recognize peptide fragments of S-antigen and mediate experimental autoimmune uveoretinitis and pinealitis. Journal of immunology (Baltimore, Md.) 136, 2875-2882 (1986).
Wacker, W.B., Donoso, L.A., Kalsow, C.M., Yankeelov, J.A., Jr. & Organisciak, D.T. Experimental allergic uveitis. Isolation, characterization, and localization of a soluble uveitopathogenic antigen from bovine retina. Journal of immunology (Baltimore, Md.) 119, 1949-1958 (1977).
Egwuagu, C.E., Mahdi, R.M., Nussenblatt, R.B., Gery, I. & Caspi, R.R. Evidence for selective accumulation of V beta 8+ T lymphocytes in experimental autoimmune uveoretinitis induced with two different retinal antigens. J Immunol 151, 1627-1636. (1993).
Pepperberg, D.R., Okajima, T.L., Ripps, H., Chader, G.J. & Wiggert, B. Functional properties of interphotoreceptor retinoid-binding protein. Photochem Photobiol 54, 1057-1060 (1991).
Takase, H., et al. Induction of suppressors of cytokine signaling (SOCS) in the retina during experimental autoimmune uveitis (EAU): potential neuroprotective role of SOCS proteins. J Neuroimmunol 168, 118-127 (2005).
Yu, C.R., et al. Suppressor of cytokine signaling 3 regulates proliferation and activation of T-helper cells. J Biol Chem 278, 29752-29759 (2003).
Egwuagu, C.E., et al. Suppressors of cytokine signaling proteins are differentially expressed in Th1 and Th2 cells: implications for Th cell lineage commitment and maintenance. J Immunol 168, 3181-3187. (2002).

This research was funded by the Intramural Research Programs of the NEI and NIH. Authors thank Dr. Wei Zhu (Laboratory of Immunology, NEI, NIH) for assistance with fundoscopy.

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TH17 cells contribute to uveitis and scleritis and are inhibited by IL-27/STAT1 in the retina. (5) Chromatin immunoprecipitation

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TH17 cells contribute to uveitis and scleritis and are inhibited by IL-27/STAT1 in the retina. (5) Chromatin immunoprecipitation

Status:

Version 1

Introduction

Reagents

Equipment

Procedure

References

Acknowledgements

Associated Publications

Status:

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Privacy Policy

Terms of Service

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