PD (16 (link)) was made up of 30 g radix pulsatillae, 9 g cortex fraxini, 5 g cortex phellodendri and 12 g rhizoma coptidis (all purchased from Longhua Hospital Shanghai University of Traditional Chinese Medicine; Shanghai, China). To obtain the water-soluble extracts, the samples were extracted using ethanol, and then concentrated, dried and powdered (17 (link)). The samples extracted by alcohol were autoclaved and stored at 4°C. The mice were randomly divided into the following five groups, with 10 mice in each group: i) PD group; ii) oxazolone-induced colitis group; iii) IL-13 (1 mg/kg body weight) intervention using anti IL-13 (PeproTech; cat. no. 500-P178) group; iv) 5-ASA (cat. no. 461814-25G; Sigma-Aldrich; Merck KGaA; 20 mg/g body weight) positive control group; and v) negative control group (equal volume saline gavage). The PD extracts (20 mg/g body weight) were administered intragastrically to the oxazolone-induced colitis mice once a day for 7 days.
Anti il 13
Manufactured by Thermo Fisher Scientific
Sourced in United States, Austria
Anti-IL-13 is a laboratory product used for research purposes. It is an antibody that binds to the cytokine interleukin-13 (IL-13). Anti-IL-13 can be utilized in various experimental techniques to study the role and function of IL-13 in biological processes.
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Lab products found in correlation
Anti cd45, by BioLegend (3 mentions)
Anti il 4, by BioLegend (3 mentions)
Anti il 5, by Thermo Fisher Scientific (2 mentions)
Anti cd103, by BD (2 mentions)
Anti cd90, by BioLegend (2 mentions)
Anti cd11c, by BD (2 mentions)
Rmil 7, by BioLegend (2 mentions)
Control rigg1, by BioLegend (2 mentions)
Rmil 2, by BioLegend (2 mentions)
Soluble anti cd28, by BioXCell (2 mentions)
Cell stimulation cocktail, by Thermo Fisher Scientific (2 mentions)
Celltrace violet cell proliferation kit, by Thermo Fisher Scientific (2 mentions)
Anti cd3ε, by BioLegend (2 mentions)
Rmil 25, by Johnson & Johnson (2 mentions)
Hybri care medium atcc 46 x, by American Type Culture Collection (1 mentions)
Pgn from staphylococcus aureus, by Merck Group (1 mentions)
Anti tnf α, by Thermo Fisher Scientific (1 mentions)
Il 13, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Anti cd25, by Thermo Fisher Scientific (1 mentions)
12 protocols using anti il 13
1
Anti-inflammatory Effects of PD Extract
2
Effects of PGN and Radiation on Colorectal Cancer
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CT26.WT is a BALB/c colon carcinoma cell line that was maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS). FHs 74. Int, a normal human intestinal cell line, was purchased from ATCC and was cultured in Hybri-Care Medium ATCC 46-X supplemented with 30 ng/ml epidermal growth factor (EGF, Peprotech, NJ, USA) and 10% FBS. HCT116 is a human colorectal carcinoma cell and was cultured in DMEM medium supplemented with 10% FBS. All cells were incubated at 37°C and 5% CO2. Both FHs 74. Int and HCT116 cells were treated with 10, 20, and 40 μg/mL PGN (from Staphylococcus aureus, Sigma, St. Louis, MO, USA). PGN was used at 20 μg/mL in all other in vitro experiments. HCT116 cells were treated with 20 μg/mL of PGN alone, 15 Gy irradiation alone, 15 Gy irradiation followed by 20 μg/mL PGN at 24 h, 15 Gy irradiation followed by 0.8 or 1.2 ng/mL IL13 (Peprotech) 2 h prior to 20 μg/mL of PGN at 24 h, 15 Gy irradiation followed by 0.12 or 0.2 μg/mL anti-IL13 (Peprotech) 2 h prior to 20 μg/mL of PGN at 24 h, or 15 Gy irradiation followed by 0.04 or 0.08 μg/mL anti-TNFα (Peprotech) 2 h prior to 20 μg/mL of PGN at 24 h.
3
Detecting Lung ILC2s and Eosinophil CD69 Expression
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To detect type 2 innate lymphoid cells (ILC2s) in the lung, cells were washed with ice-cold fluorescence-activated cell sorter (FACS) buffer (PBS containing 1% bovine serum albumin (BSA) and 1 mM EDTA), fixed in 4% paraformaldehyde, and subsequently stained with the following antibodies for 30 min at RT: anti-lineage marker cocktail (BD Biosciences), anti-CD45 (BioLegend, San Diego, CA, USA), anti-CD25 (eBioscience), anti-CD90.2 (eBioscience), anti-ST2 (eBioscience), anti-IL-5 (eBioscience) and anti-IL-13 (eBioscience). To stain for cellular CD69 in human eosinophils, the cells were stained with FITC-conjugated anti-CD69 (BioLegend) antibodies or isotype controls (BioLegend). The cells were analyzed using a FACS Canto II flow cytometer (BD Biosciences). The data were analyzed by FlowJo software version 10.6.0 (FlowJo).
4
Cytokine Analysis of Stimulated Lung Leukocytes
5
Surface and Intracellular Staining Protocol
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For surface staining, fluorochrome-conjugated antibodies were purchased from eBioscience, BioLegend, Tonbo Biosciences, and R&D Biosciences. Anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-CD25 (PC61.5), anti-GITR (DTA-1), anti-ICOS (C398.4A), anti-CTLA4 (UC10-4B9), anti-Ki67 (16A8), anti-CD62L (MEL-14), and anti-CD44 (IM7) were used in the study. For intracellular staining, surface-stained cells were fixed and permeabilized with a Foxp3 staining kit (eBioscience) according to the manufacturer’s instructions and were stained with the following antibodies: anti-Foxp3 (FJK-16s), anti-IFNg (XMG1.2), anti–IL-17A (eBio17B7), anti–IL-4 (11B11), and anti–IL-13 (eBio13A) wherever applicable. Stained cells were analyzed using an LSRFortessa SORP flow cytometer with Data-Interpolating Variational Analysis software (BD Biosciences) and FlowJo (TreeStar).
6
Comprehensive Immune Cell Profiling by Flow Cytometry
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Cell surface staining were performed with the following antibodies: anti-CD3ε, and anti-CD4, anti-CD11b, anti-CD11c, anti-CD19, anti-B220, and anti-Ly-6G (TONBO Bioscience, San Diego, CA, USA); anti-IL-33R (ST2), anti-TCRγδ, anti-CD90.2, and anti-CD45 (BioLegend, San Diego, CA, USA); anti-TCRβ, anti-CD25, anti-CD44, anti-CD62L, anti-CD69, anti-CD103, anti-NK1.1, and anti-TER119 (BD Bioscience); and anti-CD127 (eBioscience, San Diego, CA, USA). The same procedures were conducted with the following antibodies for the intracellular staining: anti-IL-4 and anti-IL-5 (BD Bioscience); and anti-IL-13 (eBioscience). Fixable Viability Dye (eBioscience) was used to exclude dead cells. All samples were analyzed using the FACS Verse (BD Bioscience) and Flow Jo software programs (Tree Star Inc., San Carlos, CA, USA).
7
Flow Cytometry Analysis of Immune Cells
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Cells were suspended in PBS supplemented with 10% FBS and 0.02% sodium azide for cell surface staining. Cells were stained with: anti-CD103, anti-CD11c, anti-CD86 (BD Biosciences, Pharmingen, San Diego, CA, USA), anti-CD90.2, anti-CD4, anti-CD8α, anti-MHC-II, anti-XCR1 (BioLegend, San Diego, CA, USA), anti-NK1.1, anti-CD19 (Ablab, Vancouver, B.C., Canada), anti-CD11b (eBioscience, San Diego, CA, USA). For intracellular staining, cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (eBioscience). Antibodies used were: anti-IL-13 (eBioscience), anti-IFNg, and anti-IL-17A (BioLegend).
8
Cytokine Profiling with Antibody Panel
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Anti-CD11c (HL3), anti-IL-4 (11B11), anti-IL-5 (TRFK5), anti-IL-10 (JES5-16E3), anti-IL-12p70 (C15.6) and anti-IL-17 (TC11-18H10) were from BD Pharmingen (Allschwil, Switzerland). Anti-IL-13 was from eBioscience (eBio13A) (Vienna, Austria).
CT was from List Biological Labs (Campbell, CA, USA). BLG was from Sigma (Buchs, Switzerland).
RPMI 1640 and DMEM medium were supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mMl -glutamine, 100 μg/ml gentamicin, 15 mM HEPES pH 7.4 and 10 % heat-inactivated FCS. In addition, DMEM was supplemented 2 × 10−5 M 2-mercaptoethanol, 1 % nonessential amino acids and 1 mM sodium pyruvate (all reagents from Sigma).
CT was from List Biological Labs (Campbell, CA, USA). BLG was from Sigma (Buchs, Switzerland).
RPMI 1640 and DMEM medium were supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM
9
ILC2-Mediated Modulation of Tumor M-MDSCs
10
ILC2-Mediated Modulation of Tumor M-MDSCs
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Intestinal ILC2s were sorted and cultured in complete RPMI (RPMI-1640 + 10% FCS + penicillin/streptomycin + 2-mercaptoethanol) with 10 ng/mL rm-IL-2 (Biolegend, 575406), 10 ng/mL rm-IL-7 (Biolegend, 577806), and 20 ng/mL rm-IL-25 (Janssen) for 3 days at 37°C, and supernatant collected (ILC2-SNT). ILC2-SNT were incubated with anti-IL-4 (Biolegend, 504122) and anti-IL-13 (eBioscience, 16-7135-85) neutralizing antibodies (αIL-4/13Ab-ILC2-SNT), or control rIgG1 (Biolegend, 400432 and eBioscience, 16-4301-85) antibodies (conAb-ILC2-SNT) for 1 hour on ice. Tumor M-MDSCs were sorted and incubated with αIL-4/13Ab-ILC2-SNT or conAb-ILC2-SNT for 2 hours at 37°C. Subsequently, sorted splenic CD8+ T cells (Live CD45+ TCRγδ-CD4-CD8+) stained with the CellTrace Violet Cell Proliferation Kit (ThermoFisher, C34557), were added to the ILC2-SNT-M-MDSC culture, and stimulated for 3 days with plate-coated anti-CD3ε (Biolegend, 100360) and 2 μg/mL soluble anti-CD28 (Bio X Cell, BE0015-1) at 37°C. Cell Stimulation Cocktail (eBioscience, 00-4975-93) was added for the last 4 hours of culture, before antibody staining. For Arginase 1 expression analysis in ILC2-SNT-treated M-MDSCs, tumor or splenic M-MDSCs were sorted and cultured with αIL-4/13Ab-ILC2-SNT or conAb-ILC2-SNT for 2 days, and stained with antibodies for flow cytometry.
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