BAL was performed on day 15 of protocol using a previously described protocol [21 (link)]. Mice were euthanized using pentobarbital sodium (65mg/kg). The lungs were lavaged with 3 volumes (1ml each time) of cold saline. Collected BAL was then kept on ice for total cell count and differential cell count. BAL was centrifuged at 1800 rpm at 4° C for 8 minutes. Supernatant was carefully collected and cryopreserved in liquid nitrogen and transferred to 80° C for further experiments. The cell pellet was resuspended with normal saline and mixed thoroughly to make a cell suspension. 10 ¼l of this suspension was mixed with of 0.4% trypan blue and a hemocytometer was used to count the viable cells. The remaining cell-pellet suspension was used for the differential cell count. The cell suspension was centrifuged at 800 rpm for 5min onto poly lysine coated glass slides. Slides were left to dry overnight at room temperature and then stained using Shandon Kwik-Diff set (Thermo Fisher Scientific, USA). Differential count of 300 cells was done per slide and then data was calculated as a percentage of total cell count. ELISA analysis was performed on the BAL supernatant using kits for Th2 cytokines IL-5 (Invitrogen, USA) and IL-13 (Millipore Sigma, USA). The plates were prepared as per the manufacturers’ instructions and absorbance was measured in plate reader at 450nm.
Il 13
Manufactured by Merck Group
Sourced in United States, Germany
IL-13 is a laboratory reagent used in research and development applications. It is a recombinant human interleukin-13 protein. Interleukin-13 is a cytokine involved in various immune and inflammatory processes. This product is intended for research use only and not for use in diagnostic or therapeutic procedures.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 4 (il 4), by Merck Group (15 mentions)
Ifn γ, by Merck Group (7 mentions)
Phorbol myristate acetate (pma), by Merck Group (6 mentions)
Lipopolysaccharides (lps), by Merck Group (5 mentions)
Il 1β, by Merck Group (4 mentions)
Tnf α, by Merck Group (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Il 18, by Merck Group (2 mentions)
Interleukin 6 (il 6), by Merck Group (2 mentions)
Il 17, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ly294002, by Merck Group (2 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (2 mentions)
Il 13, by Thermo Fisher Scientific (2 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Tgf β, by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Rnalater solution, by Thermo Fisher Scientific (1 mentions)
32 protocols using il 13
1
Bronchoalveolar Lavage Cell Analysis
2
Dolphin Lymphocyte Immune Responses
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In order to assess ability of dolphin lymphocytes to respond to a Th1, Th2, or Treg stimulus, dolphin PBMCs (2 × 106 cells/ml) were incubated in 96 well flat bottom plates with human recombinant cytokines at concentrations of 0 (unstimulated), 1, 10, and 25 pg/ml for 24 h. To assess a Th1 response, cells were stimulated with IL-12 (Millipore Sigma, Burlington, MA 01803, USA) and IFNγ (Thermo Fisher Scientific, Grand Island, NY 14072, USA) and analyzed for IFNγ expression. To stimulate a Th2 response, cells were stimulated with IL-4 (Millipore Sigma, Burlington, MA 01803, USA) and analyzed for IL-4 and IL-13 expression. To assess a Treg response, cells were stimulated with IL-2 (Thermo Fisher Scientific, Grand Island, NY 14072, USA) and TGFβ (Thermo Fisher Scientific, Grand Island, NY 14072, USA) and analyzed for TGFβ and IL-10 expression. After a 24 h incubation at 37°C and 5% CO2, cells were collected from the plates, centrifuged at 220 g for 10 min, re-suspended in RNAlater solution (Thermo Fisher Scientific, Grand Island, NY 14072, USA) and stored at 4°C for up to 1 month. RNAlater samples were then moved to −20°C for long-term storage.
3
Inflammatory Cell Quantification and Cytokine Analysis
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Total inflammatory cell numbers, including white cells and eosinophil cells were assessed by counting cells with a hemocytometer. Cytokines and enzymes including TrkA (cat. no. DG30689M; Beijing Dongge Biotechnology Co., Ltd., Beijing, China), NGF (cat. no. YM-QX2878; Shanghai YuanMu Biological Technology Co., Ltd., Shanghai, China), IgE (cat. no. RAB0799; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), IL-13 (cat. no. RAB0257; Sigma-Aldrich; Merck KGaA) and IL-18 (cat. no. BMS618-3; Thermo Fisher Scientific, Inc., Waltham, MA, USA) were determined by using the commercial ELISA kits according to the manufacturer's protocol.
4
Quantification of Inflammatory Mediators
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Concentrations of inflammatory mediators were determined in the plasma and CSF samples of all 107 subjects, as previously reported [43 (link)]. A multiplex bead array quantified concentrations of IFNγ, IL-1β, IL-4, IL-6, IL-10, IL-13, IL-17, TNFα, CCL2/MCP-1, CXCL10/IP-10, IGFBP1, and IGFBP2 (EMD Millipore, Billerica, MA, USA). Samples were diluted until the final concentrations were within the linear range of detection for the assay. Progranulin was measured by ELISA (DY2420, R&D Systems), as reported [43 (link)]. All samples were tested in duplicate and concentrations interpolated from a standard curve constructed by four-parameter fitting of internal standards.
5
Multiplexed Cytokine Profiling in Plasma
6
Primary Human Nasal Epithelial Cell Culture
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Primary human nasal epithelial cells (HNECs) were purchased from iCell Bioscience (Shanghai, China), and cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 1% penicillin (Invitrogen, USA) and 1% streptomycin (Invitrogen, USA) maintained at 37°C and 5% CO2. 50 ng/ml IL-13 (Sigma-Aldrich, USA) was used to treat HNECs for 24 h to establish an in vitro model of AR as described previously [17 (link)].
7
In Vitro Model of Allergic Rhinitis
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Primary HNECs (cat. no. HUM-iCell-m018) and 293T cell lines (cat. no. ACS-4500) were purchased from iCell Bioscience, Inc., and the American Type Culture Collection, respectively. All cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fischer Scientific, Inc.), 1% penicillin (Invitrogen; Thermo Fisher Scientific, Inc.) and 1% streptomycin (Invitrogen; Thermo Fischer Scientific, Inc.), and maintained at 37°C and 5% CO2. To establish an in vitro model of AR, HNECs were treated with 50 ng/ml IL-13 (Sigma-Aldrich; Merck KGaA) at 37°C for 24 h, as previously described (31 (link)). The control group consisted of untreated cells.
8
Human BSMC IL-13 Stimulation Assay
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Human BSMCs were obtained from Lonza (Walkersville, MD, USA). The M199 culture medium added with 10% new born calf serum (ThermoFisher Scientific, Carlsbad, CA, USA), 0.5 µg/L epidermal growth factor, and 2 µg/L fibroblast growth factor was employed for cell cultivation in a humidified 5% CO2 atmosphere at 37℃. Once cell growth reached 70%–80% confluence, it was stopped for 48 hours by transfer in serum-free media containing a 50:50 mix of F12/DMEM enhanced with 10 mL/L ITS Premix (BD Bioscience, Bedford, MA, USA). Cells were then treated with different concentrations of IL-13 for 24 hours or left untreated. IL-13 was purchased from Sigma-Aldrich (St. Louis, MO, USA).
9
Differentiation of THP-1 Macrophages
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THP-1 cells were initially derived from the European Collection of Authenticated Cell Cultures and kindly donated by the thoracic surgery department of the university clinic in Magdeburg. Cells in a density of 1 × 105 were seeded in each well of a 12-well plate with coverslips and incubated for one hour with a THP-1 medium consisting of RPMI-1640 with 2 mM of L-glutamine (Gibco, Darmstadt, Germany, 21875034) + 10% FCS (Bio&Sell, Feucht bei Nürnberg, Germany, FBS.S0615) + 1% PenStrep (Sigma-Aldrich, Taufkirchen, Germany, P0781). Phorbol-12-myristat-13-acetat (PMA, Sigma-Aldrich, Taufkirchen, Germany, P1585) with a working concentration of 50 ng/mL was added and incubated for 24 h. After 24 h, the PMA-containing medium was removed and the cells were washed with the THP-1 medium three times. Then, the THP-1 medium was added and incubated for 1 h. For differentiation in the M0, M1, and M2 macrophages, different mediums were used as follows: M0 (THP-1 medium), M1 (THP-1 medium + IFN-y (50 ng/mL), Sigma-Aldrich, Taufkirchen, Germany, SRP3058) and M2 (THP-1 medium + IL4 (Sigma-Aldrich, Taufkirchen, Germany, SRP4137) + IL13 (Sigma-Aldrich, Taufkirchen, Germany, SRP3274) (each 25 ng/mL)). Differentiation in the cells was achieved after 48 h [24 (link)].
10
M2-like Macrophage Differentiation Protocol
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THP-1 cells (1×106/well) were cultured in 6-well plates. To generate M2-like TAMs, 320 nM PMA (Sigma-Aldrich; Merck KGaA) was used to treat THP-1 cells for 6 h, and then the cells were treated with PMA plus 20 ng/ml IL-4 (Sigma-Aldrich; Merck KGaA) and 20 ng/ml IL-13 (Sigma-Aldrich; Merck KGaA) for another 18 h (24 (link)).
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