Recombinant murine il 5

Manufactured by Thermo Fisher Scientific

Recombinant murine IL-5 is a purified cytokine produced in E. coli. It functions as a growth and differentiation factor for eosinophils.

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Murine IL-5 (7 citations) Recombinant IL-5 (5 citations)

5 protocols using recombinant murine il 5

Quantifying IL-5 and IL-13 Secretion

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The secretion of IL-5 and IL-13 was assessed by enzyme-linked immunosorbent assays (ELISA) according to the manufacturer protocol using as standards recombinant murine IL-5 (PeproTech #215-15) and recombinant murine IL-13 (PeproTech #210-13).

de Lucía Finkel P., Sherwood C., Saranchova I., Xia W., Munro L., Pfeifer C.G., Piret J.M, & Jefferies W.A. (2021). Serum free culture for the expansion and study of type 2 innate lymphoid cells. Scientific Reports, 11, 12233.

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Chemotaxis Assay for Eosinophil Migration

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A chemotaxis assay was performed in Transwell plates (Corning, Corning, NY, USA) using a 5.0-μm pore size polycarbonate membrane. Various concentrations (0.1, 1 and 10 ng/mL; R&D Systems) of murine IL-17A and 100 or 500 ng/mL of murine CCL7 (Peprotech) proteins were diluted in 0.6ml of medium containing 10 ng/ml recombinant murine IL-5 (Peprotech). The medium was placed in the lower wells of the plates, and the bmEos (100 μL, 5×10⁶ /mL) were placed in the upper chambers. Cells were incubated at 37°C to permit migration across the membrane in response to CCL7 with or without IL-17A. After 3 h, the upper chambers were removed, and the number of cells that migrated to the bottom chamber in 1 min was counted using the FACSCalibur flow cytometer.

Zhang Y.L., Han D.H., Kim D.Y., Lee C.H, & Rhee C.S. (2017). Role of Interleukin-17A on the Chemotactic Responses to CCL7 in a Murine Allergic Rhinitis Model. PLoS ONE, 12(1), e0169353.

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Induction of Class-Switched Ig Isotypes

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CH12 cells were stimulated to undergo CSR to IgA by treatment with 1–5 µg/ml αCD40 (BioLegend), 5 ng/ml TGFβ (R&D Systems), and 5 ng/ml mouse recombinant IL-4 for 48 h. B lymphocytes were stimulated to undergo class switching with 5 µg/ml LPS (Sigma-Aldrich) and 5 ng/ml mouse recombinant IL-4 (Sigma-Aldrich) for CSR to IgG1; 5 µg/ml LPS only for CSR to IgG3; 5 µg/ml LPS, 10 ng/ml BAFF (PeproTech), and 2 ng/ml TGFβ for CSR to IgG2b; or 5 µg/ml LPS, 10 ng/ml BAFF, 2 ng/ml TGFβ, and 1.5 ng/ml recombinant murine IL-5 (PeproTech) for CSR to IgA. For class switching analysis, cell suspensions were stained with fluorochrome-conjugated anti-IgG1, anti-IgG3 (BD Biosciences), anti-IgG2b (BioLegend), or anti-IgA (Southern Biotech).

Delgado-Benito V., Berruezo-Llacuna M., Altwasser R., Winkler W., Sundaravinayagam D., Balasubramanian S., Caganova M., Graf R., Rahjouei A., Henke M.T., Driesner M., Keller L., Prigione A., Janz M., Akalin A, & Di Virgilio M. (2020). PDGFA-associated protein 1 protects mature B lymphocytes from stress-induced cell death and promotes antibody gene diversification. The Journal of Experimental Medicine, 217(10), e20200137.

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Clonal analysis of mouse hematopoietic stem cells

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Freshly collected mouse BM was labeled according to panels exemplified in Supplementary Data. Single HSCs (LSK⁺CD34⁻Flt3⁻CD150⁺CD48⁻) were sorted into 96-well plates, using an Aria FACS Fusion II with a 100-μm nozzle. Cells were cultured for 10 days in IMDM-10% (see the “Cell culture” section), supplemented with SCF (50 ng/ml; recombinant murine SCF, PeproTech, #250-03), interleukin-3 (IL-3; 10 ng/ml; recombinant murine IL-3, PeproTech, #213-13), IL-6 (10 ng/ml; recombinant murine IL-6, PeproTech, #216-16), erythropoietin (EPO; 2 U/ml; BioLegend, #587602), IL-11 (50 ng/ml; recombinant murine IL-11, PeproTech, #220-11), IL-5 (10 ng/ml; recombinant murine IL-5, PeproTech, #215-15), thrombopoietin (TPO; 50 ng/ml; recombinant murine TPO, PeproTech, #315-14), IL-4 (10 ng/ml; recombinant murine IL-4, PeproTech, #214-14), granulocyte-macrophage colony-stimulating factor (GM-CSF; 15 ng/ml; recombinant murine GM-CSF, PeproTech, #315-03), and IL-7 (10 ng/ml; recombinant murine IL-7, PeproTech, #217-17). Growth factors/cytokines were refreshed at day 5. The clones/colonies in each well were labeled on day 10 with anti-CD117, anti-Ter119, anti-CD11b, and anti-IgD antibody. Clones were analyzed by flow cytometry using the BD Fortessa II (BD Biosciences) and processed with FlowJo 10.v.

Pessoa Rodrigues C., Herman J.S., Herquel B., Valsecchi C.I., Stehle T., Grün D, & Akhtar A. (2020). Temporal expression of MOF acetyltransferase primes transcription factor networks for erythroid fate. Science Advances, 6(21), eaaz4815.

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Induction of Class-Switch Recombination

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CH12 cells were stimulated to undergo CSR to IgA by treatment with 1-5 µg/mL aCD40 (BioLegend), 5 ng/ml TGFb (R&D Systems) and 5 ng/ml of mouse recombinant IL-4 for 48 h. B lymphocytes were stimulated to undergo class switching with 5 µg/ml LPS (Sigma-Aldrich) and 5 ng/ml of mouse recombinant IL-4 (Sigma-Aldrich) for CSR to IgG1; 5 µg/ml LPS only for CSR to IgG3; 5 µg/ml LPS, 10 ng/ml BAFF (PeproTech) and 2 ng/ml TGFb for CSR to IgG2b; or 5 µg/ml LPS, 10 ng/ml BAFF, 2 ng/ml TGFb and 1.5 ng/ml recombinant murine IL-5 (PeproTech) for CSR to IgA. For class switching analysis, cell suspensions were stained with fluorochrome-conjugated anti-IgG1, anti-IgG3 (BD-Biosciences), anti-IgG2b (BioLegend), or anti-IgA (Southern Biotech).

Delgado-Benito V., Berruezo-Llacuna M., Altwasser R., Winkler W., Balasubramanian S., Sundaravinayagam D., Graf R., Rahjouei A., Driesner M., Jung L.A., Janz M., Akalin A., & Virgilio M.D. (2020). Pdap1 protects mature B lymphocytes from stress-induced cell death and promotes antibody gene diversification.

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