Mouse intestinal carcinogenic epithelial cells (CMT93) obtained from ATCC were cultured in high-glucose DMEM medium (Gibco) containing 10% FBS, 1x MEM and 5mM HEPES and 50U/ml penicillin-streptomycin. 5x104 cells/well were grown on sterile glass coverslips in a 12-well plate (Corning) in a humid incubator at 37°C and 5% CO2. After reaching 50% confluency, the cells were treated for 24hr with 100ng/ml recombinant mouse IL-22 (R&D Biosystems) or RELM-β (Peprotech). Cells were subsequently fixed in ice-cold paraformaldehyde for 15 min followed by blocking with 1% BSA for 30min and incubation with rabbit derived anti-Ki67 (Thermo Scientific) for 2hr. After washing 3X with PBS, cells were incubated in dark for 1hr with secondary antibody conjugated to Alexafluor 568. After removal of secondary antibodies, cells were washed 3X with PBS followed by mounting in Vectashield (Vector Laboratories, Burlington, ON, Canada) on glass slides and screened with a Zeiss microscope.
Recombinant mouse il 22
Manufactured by R&D Systems
Sourced in United States
Recombinant mouse IL-22 is a cytokine that is produced by various immune cells, including T cells, NK cells, and innate lymphoid cells. It plays a role in the regulation of immune responses and tissue homeostasis.
Automatically generated - may contain errors
Lab products found in correlation
12 well plate, by Corning (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Dmem high glucose medium, by Thermo Fisher Scientific (1 mentions)
Relm β, by Thermo Fisher Scientific (1 mentions)
Lgr5 egfp ires creert2 mice, by Jackson ImmunoResearch (1 mentions)
Acetaldehyde, by Tianjin Damao (1 mentions)
Il22jop, by Thermo Fisher Scientific (1 mentions)
Ebiomm17f3, by Thermo Fisher Scientific (1 mentions)
Cmt 93, by American Type Culture Collection (1 mentions)
Recombinant mouse il 17a, by R&D Systems (1 mentions)
Af582, by R&D Systems (1 mentions)
4 protocols using recombinant mouse il 22
1
IL-22 and RELM-β Regulate Intestinal Cell Proliferation
2
Lgr5-EGFP-IRES-CreERT2 Intestinal Organoid Assay
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Lgr5-EGFP-IRES-creERT2 mice (Stock no. 008875) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and a total of 5 male, Lgr5-EGFP-IRES-creERT2 mice (8 weeks old, weighing approximately 20 g) were kept under SPF experiment animal conditions (temperature,23±2°C; humidity, 65±5%) in a 12-h light/dark cycle and fed a standard laboratory diet and water in East China Normal University Center for Animal Research. Endogenous GFP (green) driven by Lgr5 promoter indicates crypt base columnar stem cells in the small intestine. Crypts from the Lgr5-EGFP-IRES- CreERT2 mice were isolated as described above after the mice were sacrificed and organoid culture was performed. Following treatment with 100 ng/ml recombinant mouse IL-22 (R&D Systems, Inc., Minneapolis, MN, USA), organoids were collected and dissociated with TypLE and 2,000 U/ml DNase for 15 min at 37°C. The dead cells were then excluded by scatter characteristics and propidium iodide. Lgr5+ISCs were identified by their endogenous GFP expression.
3
Acetaldehyde-Induced Oxidative Stress in HSC-T6 Cells
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HSC-T6 cells were purchased from the cell bank of the Central South University (Hunan, China). Acetaldehyde (40%) was purchased from Tianjin DaMao Chemical Reagents (Tianjin, China). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Sijiqing (Hangzhou, China). Fetal calf serum (10%) was purchased from Hyclone (Logan, UT, United States). Recombinant mouse IL-22 was purchased from R&D Systems (Minneapolis, MN, United States). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit and nuclear-cytoplasmic protein extraction kit were purchased from Boster Bioengineering Company (Wuhan, China). Malondialdehyde (MDA) and GSH kits were purchased from Jiancheng Company (Nanjing, China). α-smooth muscle actin (SMA) and Nrf2 polyclonal antibody were purchased from Abcam (Cambridge, MA, United States). Horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibodies were purchased from Boster Bioengineering.
4
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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