Six- to 8-week-old male C57BL/6 mice (Charles River Laboratory, Wilmington, MA) and IL-22 KO mice (UC Davis MMRRC, Davis, CA) were used in accordance with the standards of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The research protocol was approved by the Yonsei University College of Medicine. The in vivo mouse experiments were performed at the Institute of Vision Research at the Yonsei University College of Medicine. DED was induced in mice as described previously.6 (link), 7 (link), 39 (link), 40 (link) In brief, mice were exposed to a controlled environment chamber (CEC), which allows the controlled regulation and maintenance of the temperature (21 – 23 °C), relative humidity (< 30 %), and airflow (15 L/min). Clinical signs such as corneal erosions were assessed by corneal fluorescein staining using 1% fluorescein (Sigma-Aldrich, St. Louis, MO) according to the standard National Eye Institute scoring system. One week post DED induction, mice were euthanized and tissues were collected for immunohistochemistry and molecular studies. For in vivo IL-22 blockade experiments, 100 μg/mouse of αIL-22 (AF582, R&D Systems, Minneapolis, MN) or PBS were intravenously injected into WT mouse at 2-hour before placing them into the CEC and then daily for 3 days.
Af582
Manufactured by R&D Systems
Sourced in United States
AF582 is a laboratory reagent manufactured by R&D Systems. It functions as a recombinant human protein.
Automatically generated - may contain errors
Lab products found in correlation
C57bl 6 mice, by Charles River Laboratories (2 mentions)
Murine tnf α, by Thermo Fisher Scientific (1 mentions)
Ab 108 c, by R&D Systems (1 mentions)
Cmt 93, by American Type Culture Collection (1 mentions)
Recombinant mouse il 17a, by R&D Systems (1 mentions)
Ebiomm17f3, by Thermo Fisher Scientific (1 mentions)
Il22jop, by Thermo Fisher Scientific (1 mentions)
Recombinant mouse il 22, by R&D Systems (1 mentions)
5 protocols using af582
1
Dry Eye Disease Induction in Mice
2
Intestinal Stem Cell Coculture Assay
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For coculture experiments, the isolated LPMCs were cultured with intestinal crypts at a ratio of 20:1 in Matrigel according to previous work, though with a slight modification [12 (link)]. The effective concentration of L-fucose on ISC proliferation was selected in the concentration gradient experiment (Figure S1 ). L-fucose (5 mg/mL) and murine anti-IL-22 neutralizing antibody (0.1 μg/mL, AF582, R&D system, Minneapolis, MN, USA) were added independently or simultaneously to the culture medium for 5 days, and the medium was changed every two days. Murine TNF-α (60 ng/mL, Peprotech, Rocky Hill, NJ, USA) was used to induce the inflammatory model for 24 h.
3
Intradermal IL-23 and IL-22 Neutralization
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Control or neoATB mice were intradermally injected with recombinant IL-23 (1 μg per day) at opposing sides of shaved back skin (0.5 μg on each side) for 6 consecutive days. For experiments requiring neutralization of local production of IL-22, neoATB mice were intradermally injected at day 1 (50 μg) and day 3 (15 μg) with neutralizing IL-22 antibody (R&D, AF582) during a 4-day imiquimod treatment. Control mice were intradermally injected at day 1 (50 μg) and day 3 (15 μg) with control goat IgG (R&D, AB-108-C) antibody instead.
4
Dry Eye Disease Induction in Mice
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Six- to 8-week-old male C57BL/6 mice (Charles River Laboratory, Wilmington, MA) and IL-22 KO mice (UC Davis MMRRC, Davis, CA) were used in accordance with the standards of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The research protocol was approved by the Yonsei University College of Medicine. The in vivo mouse experiments were performed at the Institute of Vision Research at the Yonsei University College of Medicine. DED was induced in mice as described previously.6 (link), 7 (link), 39 (link), 40 (link) In brief, mice were exposed to a controlled environment chamber (CEC), which allows the controlled regulation and maintenance of the temperature (21 – 23 °C), relative humidity (< 30 %), and airflow (15 L/min). Clinical signs such as corneal erosions were assessed by corneal fluorescein staining using 1% fluorescein (Sigma-Aldrich, St. Louis, MO) according to the standard National Eye Institute scoring system. One week post DED induction, mice were euthanized and tissues were collected for immunohistochemistry and molecular studies. For in vivo IL-22 blockade experiments, 100 μg/mouse of αIL-22 (AF582, R&D Systems, Minneapolis, MN) or PBS were intravenously injected into WT mouse at 2-hour before placing them into the CEC and then daily for 3 days.
5
IL-22 and IL-17A Modulate Antimicrobial Peptides in Colonic Epithelial Cells
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The colonic epithelial cell line, CMT-93, was purchased from the American Type Culture Collection and used between passages 1 to 3. CMT-93 cells were cultured until 80% confluency in DMEM with 10% FBS. Antimicrobial peptide expression was measured after incubation with IL-22 and IL-17A. Cells were treated with recombinant mouse IL-22 (10 ng/ml; R&D Systems) or recombinant mouse IL-17A (50 ng/ml; R&D Systems) or both for 48 hours in full medium. Conditioned medium from Th17-polarized T cells treated with or without 10 μM PY109 was collected after 4 days, frozen in aliquots, and stored at −80°C. CMT-93 cells were incubated with a 1:1 mix of conditioned medium and DMEM with 10% FBS for 48 hours. Th17 polarization medium in the absence of T cells with or without 10 μM PY109 served as a negative control. In select experiments, anti–IL-22 (IL-22JOP, eBioscience or AF582, R&D Systems) or anti–IL-17A (eBioMM17F3, eBioscience) neutralizing antibodies or the corresponding IgG isotype was added to culture medium at a concentration of 4 μg/ml.
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