Rmil 5

Manufactured by Thermo Fisher Scientific

Sourced in United States

The RmIL-5 is a lab equipment product designed for conducting research and analysis. It serves as a tool for researchers and scientists to study interleukin-5, a cytokine involved in various biological processes. The core function of the RmIL-5 is to enable the detection, quantification, and analysis of interleukin-5 levels in samples, supporting research and investigation in relevant fields.

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7 protocols using rmil 5

B Cell Switching and Activation

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B cells were isolated with the EasySep Mouse B cell isolation kit (STEMCELL Technologies, Canada) from splenocytes. To induce CSR from IgM to IgG1, B cells (5 × 10⁵ to 1 × 10⁶ cells/ml) were activated with LPS (25 μg/ml) from Escherichia coli O55:B5 (Sigma, St. Louis, MO) and rmIL-4 (10 ng/ml) at 37°C (5% CO₂). For IgA switching, cells were activated with anti-CD40 (1 μg/ml, clone 1C10, BioLegend), rmIL-4 (10 ng/ml, Peprotech), rmIL-5 (10 ng/ml, Peprotech), and rhTGFβ1 (transforming growth factor β 1, 1 ng/ml). Media were composed of RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS, 1× minimum essential medium (MEM) non-essential amino acids, 10 mM Hepes, 2 mM Glutamax, 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol, and gentamicin (50 μg/ml) (all from Thermo Fisher Scientific, Waltham, MA, USA). To enhance Cre^ERT2-mediated deletion, we cultured cells from Cre^ERT2 mice in the presence of 1 μM 4-hydroxytamoxifen (Tocris). All cytokines used above were from PeproTech (Rocky Hill, NJ, USA).

Lio C.W., Shukla V., Samaniego-Castruita D., González-Avalos E., Chakraborty A., Yue X., Schatz D.G., Ay F, & Rao A. (2019). TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer. Science immunology, 4(34), eaau7523.

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Optimizing B Cell Class Switching

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B cells were isolated with the EasySep Mouse B cell isolation kit (Stem Cell Technology, Canada) from total splenocytes. To induce CSR from IgM to IgG1 or IgE, B cells (5×10⁵−1×10⁶ cells/ mL) were activated with 1 μg/mL anti-CD40 clone 1C10, Biolegend) and rmIL-4 (10 ng/mL, Peprotech). For IgG3 and IgG2b switching, B cells were activated with 25 ug/mL of LPS from E. coli O55:B5 (Sigma, St. Louis, MO) and 10 ng/mL rmIL-4 at 37°C 5% CO 2. For IgA and IgG1 switching, B cells were activated with anti-CD40 (1 μg/mL, clone 1C10, Biolegend), rmIL-4 (10 ng/mL, Peprotech), rmIL-5 (10ng/mL, Peprotech), and rhTGFβ (1 ng/mL).

Shukla V., Halabelian L., Balagere S., Samaniego-Castruita D., Feldman D.E., Arrowsmith C.H., Rao A, & Aravind L. (2019). HMCES functions in the alternative end-joining pathway of the DNA DSB repair during class switch recombination in B cells.. Molecular cell, 77(2), 384-394.e4.

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Generating Bone Marrow-Derived Eosinophils

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Bone marrow-derived eosinophils (BMDEs) were generated as described (25 (link)). Briefly, bone marrow cells were collected from the femur and tibia bones by crushing and red blood cells will be lysed with ACK lysis buffer (Sigma). Low-density bone marrow progenitors were separated by gradient centrifugation (Histopaque 1083, Sigma) of 1700 RPM for 30 minutes. Low density bone marrow cells were seeded at 5 X 10⁵ cells/mL in 24 wells plate (BD Falcon) in media containing IMDM (Gibco) with 10% fetal bovine serum (HyClone), 1% penicillin-streptomycin (Biological industries), 2 mM glutamine (Gibco),50 μM β-mercaptoethanol (Sigma) and supplemented with 100 ng/mL stem-cell factor (SCF; PeproTech) and 100 ng/mL FLT3-Ligand (FLT3-L; PeproTech) from day 0 to day 4. On day 4, the media containing SCF, and FLT3-L replaced with media containing 10 ng/ml recombinant mouse interleukin-5 (rmIL-5; Peprotech) alone. Medium refreshing was done every 3 days from day 4 to 14 until eosinophil purity reached >85%. Purity was assessed by flow cytometry using CCR3 (R&D) and Siglec-F (BD Bioscience) as eosinophils markers.

Dolitzky A., Hazut I., Avlas S., Grisaru-Tal S., Itan M., Zaffran I., Levi-Schaffer F., Gerlic M, & Munitz A. (2022). Differential regulation of Type 1 and Type 2 mouse eosinophil activation by apoptotic cells. Frontiers in Immunology, 13, 1041660.

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Murine Bone Marrow-Derived Eosinophil Isolation

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Bone marrows from the femur and tibia of 6- to 8-week-old mice were flushed with PBS and processed into a single-cell suspension. After red blood cells were lysed with red blood cell lysis buffer, cells were resuspended in bone marrow medium [RPMI 1640 containing 20% FBS, 55 μM β-mercaptoethanol, 10 mM nonessential amino acids, 1 mM sodium pyruvate, penicillin (100 IU/ml), streptomycin (100 μg/ml), 2 mM glutamine, and 25 mM Hepes buffer] at a concentration of 1 × 10⁶ cells/ml. Cells were stimulated with stem cell factor (SCF; 100 ng/ml) and fms-like tyrosine kinase 3 ligand (FLT3L; 100 ng/ml) (both from PeproTech, Rocky Hill, NJ) for 4 days. On day 4, the medium containing SCF and FLT3L was replaced with a medium containing only rmIL-5 (10 ng/ml; PeproTech, Rocky Hill, NJ). On day 8, cells were transferred to new flasks and maintained in fresh medium supplemented with rmIL-5. Half of the medium was replaced with fresh medium containing rmIL-5 every other day, and the cell concentration was adjusted each time to 1 × 10⁶ cells/ml. On day 14, BMDEs were stimulated with mIFNγ (15 ng/ml) and mTNF (15 ng/ml) for 16 hours to activate cells. BMDE purity and viability were analyzed by flow cytometry.

Cheng J.N., Luo W., Sun C., Jin Z., Zeng X., Alexander P.B., Gong Z., Xia X., Ding X., Xu S., Zou P., Wan Y.Y., Jia Q., Li Q.J, & Zhu B. (2021). Radiation-induced eosinophils improve cytotoxic T lymphocyte recruitment and response to immunotherapy. Science Advances, 7(5), eabc7609.

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Isolation and Culture of Mouse Bone Marrow Cells

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BALB/c mice were sacrificed and their femurs were isolated. Femoral marrow was rinsed with RPMI 1640 medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), supplemented with streptomycin and penicillin (HyClone), 20% fetal bovine serum (HyClone), Fms-related tyrosine kinase 3 ligand (FLT3-L; PeproTech, Inc., Rocky Hill, NJ, USA), stem cell factor (SCF; PeproTech, Inc.) and recombinant mouse IL-5 (rmIL-5; PeproTech, Inc.). A total of 10⁷ cells/ml were incubated at 37°C in 5% CO₂.

Liu Y., Zhu X, & Zhang H. (2016). Effects of chemokine receptor 3 gene silencing by RNA interference on eosinophils. Experimental and Therapeutic Medicine, 13(1), 215-221.

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Eosinophil Differentiation and Cytokine Response

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Bone marrow-derived eosinophils were cultured in Iscove's modified Dulbecco's medium (IMDM; Invitrogen) with 20% FBS (Invitrogen), 100 IU/mL penicillin and 10 mg/mL streptomycin, 2 mM glutamine, 25 mM HEPES, 1 × nonessential amino acids, 1 mM sodium pyruvate and 0.006‰ β-mercaptoethanol (Sigma-Aldrich, St. Louis, USA) supplemented with 100 ng/mL rmSCF (PeproTech) and 100 ng/mL recombinant murine FLT-3 ligand (rm-FLT3-L; PeproTech) from days 0 to 4. On day 4, the medium was replaced with medium containing 10 ng/mL rmIL-5 (PeproTech). The cells were provided with fresh medium supplemented with rmIL-5 every 3 days until to 14 days. A total of 1 × 10 6 bone marrow-derived eosinophils per group were treated with 5.0 μg/cm 2 PM2.5 for 48 h with or without 100 ng/mL ruxolitinib (InvivoGen, San Diego, USA). After 48 h, the cell supernatants were harvested. The concentrations of IL-1β, IL-5 and IL-13 in the supernatants were determined using ELISA.

Yang Y., Li X., An X., Zhang L., Li X., Wang L, & Zhu G. (2020). Continuous exposure of PM2.5 exacerbates ovalbumin-induced asthma in mouse lung via a JAK-STAT6 signaling pathway. Advances in clinical and experimental medicine : official organ Wroclaw Medical University, 29(7).

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Isolation of Bone Marrow Eosinophils

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Male WT and Ox40^-/- mice were used to obtain bone marrow eosinophils. After treatment with erythrocyte lysis buffer to remove RBCs, bone marrow cells were used for culture and sorting of mature BM eosinophils. Unselected mouse bone marrow progenitors harvested from WT or Ox40^–/– mice were cultured with recombinant mouse (rm)FLT3-L (100 ng/ml, 250-31L, PeproTech, USA) and rmSCF (100 ng/ml, 250-03, PeproTech) for 4 days, followed by culture with rmIL-5 (10 ng/ml, 215-15, PeproTech) alone thereafter for 4-6 days to generate bone marrow-derived eosinophils (bmEos) as previously described (22 (link)). On Days 8-10, cultured eosinophils were purified with a FACS Aria II cell sorter (BD Biosciences, CA, USA) using gating on 7AAD-Siglec-F+SSC-A^hi cells as previously described (22 (link)). For natural BM eosinophils, mature BM eosinophils were sorted as previously described (23 (link)). Briefly, B cells (anti-B220), T cells (anti-CD3), DCs (anti-CD11c), neutrophils and macrophages (anti-Gr-1) were depleted by MACS. Then, the remaining cells were incubated with anti-7AAD, anti-FcϵRIα, anti-Siglec-F, and anti-CD11b antibodies. Live mature eosinophils, gated as 7AAD-FcϵRIα-Siglec-F+CD11bint cells, were sorted with a FACS Aria II cell sorter.

Xu L., Tian D., Zhou M., Ma J., Sun G., Jin H., Li M., Zhang D, & Wu J. (2022). OX40 Expression in Eosinophils Aggravates OVA-Induced Eosinophilic Gastroenteritis. Frontiers in Immunology, 13, 841141.

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