Recombinant murine il 7

Manufactured by R&D Systems

Recombinant murine IL-7 is a cytokine that promotes the survival and proliferation of T cells and B cells. It is produced in a mammalian cell expression system.

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5 protocols using recombinant murine il 7

Isolation and Culture of Type 2 Innate Lymphoid Cells

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Single-cell suspensions from lung and small intestine were stained with respective surface antibodies and viability dye as described above and sorted on a BD FACSAria™ III Cell Sorter (BD Biosciences) as live CD45⁺Lin⁻Thy-1⁺ST2⁺CD25⁺ or live CD45⁺Lin⁻Thy-1⁺CD127⁺KLRG1⁺ cells, respectively. Isolated cells were cultured for 18 h in complete ILC2 medium containing recombinant murine IL-7 (10 ng/ml; R&D Systems), washed, and rested for 4 h in complete ILC2 medium without cytokines before being used in experiments.

Mindt B.C., Krisna S.S., Duerr C.U., Mancini M., Richer L., Vidal S.M., Gerondakis S., Langlais D, & Fritz J.H. (2021). The NF-κB Transcription Factor c-Rel Modulates Group 2 Innate Lymphoid Cell Effector Functions and Drives Allergic Airway Inflammation. Frontiers in Immunology, 12, 664218.

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In vivo IL-7 treatment and anti-IL-7R blockade in Aspergillus fumigatus infection

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For in vivo IL-7 treatment, WT BL/6 mice were chronically exposed to A. fumigatus as described. On days 7, 9, 11 and 14, mice received 1.5 μg of carrier-free recombinant murine IL-7 (R&D Systems) (dose based on refs.^{33 (link),34 (link)}) in a volume of 50 μl intratracheally. Controls received 50 μl of diluent (PBS) intratracheally. For in vivo anti-IL-7R (CD127) blockade, WT BL/6 mice were chronically exposed to A. fumigatus as described. On days 7, 9, 11 and 14, mice received 0.5 mg of InVivoMAb rat anti-mouse IL-7Rα/CD127 (Catalog #BE0065, Bio X Cell, West Lebanon, NH) in a volume of 0.2 ml intraperitoneally. Controls received 0.5 mg of InVivoMAb Rat IgG2a Isotype control (anti Trinitrophenol; Catalog #BE0089, Bio X Cell).

Reeder K.M., Dunaway C.W., Blackburn J.P., Yu Z., Matalon S., Hastie A.T., Ampleford E.J., Meyers D.A, & Steele C. (2018). The common γ-chain cytokine IL-7 promotes immunopathogenesis during fungal asthma. Mucosal immunology, 11(5), 1352-1362.

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Cytokine-Induced IFN-γ Production

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iSell^Tomato mice were infected with AdV and CD45⁺CD62L⁻CD4⁺Tomato⁺ IE T cells were sorted 10 days post infection. Sorted T cells were cultured in RPMI 1640 (Gibco), 10% FBS (Sigma), 1% Pen/Strep (Gibco), 1% L-glutamine (Gibco), 1% Sodium Pyruvate (Gibco), 2% Non-essential Amino Acids (Gibco), 2.5% 1M HEPES (Gibco), 50μM 2-Mercaptoethanol (Sigma), 10 ng/ml recombinant murine IL-2 (R&D) and 5 ng/ml recombinant murine IL-7 (R&D) at 10–20.000 cells/well in a 96-well round bottom plate (Corning). T cells were stimulated with 10 ng/ml recombinant murine IL-12 (R&D), 10 ng/ml recombinant murine IL-15/IL-15Rα complex (ThermoFisher) and 10 ng/ml recombinant murine IL-18 (R&D). Supernatants were collected 24-hour post stimulation and IFN-γ was measured with IFN-γ ELISA (Invitrogen) by following manufacturer’s instructions.

Parsa R., London M., de Castro T.B., Reis B., des Amorie J.B., Smith J.G, & Mucida D. (2022). Newly recruited intraepithelial Ly6A+CCR9+CD4+ T cells protect against enteric viral infection. Immunity, 55(7), 1234-1249.e6.

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Murine Bone Marrow B Cell Culture

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Mice were sacrificed by cervical dislocation at 6–8wk of age and bone marrow cells were flushed from the femora and tibiae. Cells were prepared as described (29 (link)). Briefly, adherent cells were removed by incubating collected bone marrow cells at 37 °C for 15min. Non-adherent cells were collected by centrifugation, subjected to red blood cell lysis in ACK Lysing Buffer (Thermo Fisher), washed, and seeded at a density of 7.5×10⁵/mL in 1X Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% fetal bovine serum (Life Technologies), 1X penicillin/streptomycin (Corning), 2mM L-glutamine (Sigma-Aldrich), 5.5×10⁻⁵ M β-mercaptoethanol (Sigma-Aldrich), and 10 ng/μl of recombinant murine IL-7 (R&D systems). Cells were cultured at 37 °C with 5% CO₂ for two consecutive periods of 4d, with cells being re-seeded at their initial density after the first culture period. For experiments including IL-7 withdrawal, cells were washed twice in 10 mL of culture medium and re-seeded at 7.5×10⁵/ml in fresh medium containing 20 ng/μl of recombinant murine B cell activating factor (BAFF, R&D systems) and cultured 2d prior to analysis.

Schabla N.M., Perry G.A., Palmer V.L, & Swanson P.C. (2018). VprBP/DCAF1 Regulates RAG1 Expression Independently of Dicer by Mediating RAG1 Degradation. Journal of immunology (Baltimore, Md. : 1950), 201(3), 930-939.

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Transduction of Activated Murine T Cells

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Splenocytes from WT mice were activated in vitro with 1 μg/ml anti-CD3ε (145-2C11, BD Biosciences) and 1 μg/ml anti-CD28 (37.51, BD Biosciences) in the presence of 10 ng/μl recombinant human IL-2 (Peprotech) and 5 ng/μl recombinant murine IL-7 (R&D Systems) in T cell media (DMEM, 10% FBS, 2 μM L-glutamine, 100 U/ml penicillin/streptomycin, 25 μM 2-β-mercaptoethanol) at 37 °C, 5% CO₂. After 2 days, activated T cells were centrifuged and incubated with rAAV serotypes (UPenn Vector Core) engineered to express GFP. After 3 days, GFP expression in live T cells was determined by flow cytometry.

Rollins M.R., Raynor J.F., Miller E.A., Butler J.Z., Spartz E.J., Lahr W.S., You Y., Burrack A.L., Moriarity B.S., Webber B.R, & Stromnes I.M. (2023). Germline T cell receptor exchange results in physiological T cell development and function. Nature Communications, 14, 528.

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