Rmil 4

Manufactured by Thermo Fisher Scientific

Sourced in United States

RmIL-4 is a recombinant mouse interleukin-4 protein. Interleukin-4 is a cytokine that plays a crucial role in the regulation of immune responses.

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Recombinant murine interleukin (rmIL)-4 (2 citations)

44 protocols using rmil 4

MDSC Migration Assay Protocol

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MDSCs were obtained by flow cytometric sorting. MDSCs were washed with PBS twice and resuspended with 5 × 10⁵/mL in a DMEM with 10% FBS, 10 ng/mL rmIL-4 (Peprotech, USA). Transwell chambers (Corning, USA) were placed in a 24-well plate, the lower chamber was filled with DMEM with 10% FBS, 10 ng/mL rmIL-4 with/without 100 ng/mL rmG-CSF (Peprotech, USA), and the upper chamber was filled with MDSCs. After 4 h, cells were harvested in the lower chamber and counted by the cells counts.

Li W., Zhang X., Chen Y., Xie Y., Liu J., Feng Q., Wang Y., Yuan W, & Ma J. (2016). G-CSF is a key modulator of MDSC and could be a potential therapeutic target in colitis-associated colorectal cancers. Protein & Cell, 7(2), 130-140.

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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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Evaluating BMDC Activation by R837

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BMDCs obtained from C57BL/6 mice were co-cultured with R837, NS (negative control) or LPS (positive control) as previously described [20 (link)]. Briefly, BMDCs were obtained from the femur and tibia of C57BL/6 mice, and cultured with 20 ng mL⁻¹ rmGM-CSF (Xiamen Amoytop Biotech Co., Ltd., China) and 10 ng mL⁻¹ rmIL-4 (Pepro Tech, USA). The medium was replaced three-quarters every 2–3 days until adherent cells were collected on the 8th day. The harvested immature DCs were resuspended in RPMI 1640 medium without cytokines and co-cultured with R837, NS or LPS for 48 h. Then incubated with 1 µL FITC-anti-mouse CD11c monoclonal antibody, PE-anti-mouse CD86 monoclonal antibody, and APC-anti-mouse CD80 monoclonal antibody (Biolegend, USA) for 30 min before evaluated by flow cytometry (Beckmann, BD Bioscience, USA).

Liu Q., Xu R., Shen J., Tao Y., Shao J., Ke Y, & Liu B. (2024). In situ chemoimmunotherapy hydrogel elicits immunogenic cell death and evokes efficient antitumor immune response. Journal of Translational Medicine, 22, 341.

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Generating Murine Dendritic Cells from Bone Marrow

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Dendritic cells were generated from murine bone marrow cells by Inaba’s protocol^{23 (link)} with minor modifications. Briefly, bone marrow cells were flushed from the femurs of mice, followed by incubation with red blood cell (RBC) lysing buffer (Sigma-Aldrich, St Louis, Missouri) to deplete the RBCs. The cells were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, 8 ng/mL recombinant murine (rm) GM-CSF (PeproTech, Rocky Hill, New Jersey), and 2 ng/mL rmIL-4 (PeproTech). On day 1 of culturing, nonadherent cells were gently removed by gently shaking the dishes, and fresh medium was added. On day 3 of culturing, the LLC1 cell lysate prepared by 5 cycles of freezing and thawing of LLC1 cells were added into cultures, which contained 1 × 10⁶ DCs, to obtain a final concentration of 5 mg/mL protein according to the study by Thumann et al.^{24 (link)} On day 5 of culturing, the cultures were supplemented with 5 ng/mL recombinant human tumor necrosis factor-α (rmTNF-α) (PeproTech) to induce the maturation of the DCs, which were subsequently exposed to X-ray radiation at a dose of 0.2 Gy the next day. The X-rays were given using a precision linear accelerator (Elekta, Stockholm, Sweden) with a dose rate of 442.89 cGy/min.

Wang S., Yu H., He R., Song X., Chen S., Yu N., Li W., Li F, & Jiang Q. (2019). Exposure to Low-Dose Radiation Enhanced the Antitumor Effect of a Dendritic Cell Vaccine. Dose-Response, 17(1), 1559325819832144.

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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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Dendritic Cell Differentiation and Activation

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Bone marrow cells were obtained from C57BL/6 mice and were separated and prepared as single-cell suspensions; erythrocytes were lysed with ACK buffer. The residual bone marrow cells were cultured in RPMI 1640 containing 10% heat-inactivated fetal bovine serum (HyClone), 100 IU/ml penicillin (Gibco), 1% sodium pyruvate (Corning), and 1% HEPES in the presence of 50 ng/ml rm-GM-CSF (Peprotech) and 2.5 ng/ml rm-IL-4 (Peprotech) to induce the differentiation of dendritic cells. After 6 days of incubation at 37 °C and 5% CO₂, the dendritic cells were identified by using FACS staining with specific fluorescence-labeled CD11c antibody (Biolegend). The dendritic cells were incubated with ALD-DNA overnight with or without additional NaCl (20 mM, Sigma)^{31 (link),32 (link),70 (link),101 (link)} and with or without the STAT1 inhibitor fludarabine (2 μg/ml, Selleck-21679-14-1) and p38 MAPK kinase inhibitor (5 μM, InvivoGen-tlrl-sb20). The harvested dendritic cells were collected for transfer to C57BL/6 mice or for flow cytometric measurement of activation and/or maturation markers.

Xiao Z.X., Hu X., Zhang X., Chen Z., Wang J., Jin K., Cao F.L., Sun B., Bellanti J.A., Olsen N, & Zheng S.G. (2020). High salt diet accelerates the progression of murine lupus through dendritic cells via the p38 MAPK and STAT1 signaling pathways. Signal Transduction and Targeted Therapy, 5, 34.

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Murine Myeloid Progenitor Enrichment

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Bone marrow was collected by aspiration from mouse femurs and tibias and processed to generate single-cell suspensions. After red blood cell lysis, progenitors were enriched by negative selection by staining with biotinylated anti-CD4 (RM4-5; BD), anti-CD5 (53-7.3; BD), anti-CD8α (53-6.7; BD), anti-CD19 (1D3; BD), and anti-B220 (RA3-6B2; BD) followed by magnetic depletion with antibiotin microbeads (#130-090-485; Miltenyi Biotec). Enriched progenitors were plated at 5 × 10⁵ cells/well in 24-well plates and cultured in RPMI supplemented with 10% FCS, penicillin/streptomycin, 10 mM Hepes, 2-mercaptoethanol, 10 ng/ml recombinant murine GM-CSF (Peprotech), and 5 ng/ml rmIL-4 (Peprotech). Every other day, half the medium was removed and replaced with fresh differentiation medium. After 6 d of culture, repeated pipetting was used to collect loosely adherent cells for analysis.

Vander Lugt B., Riddell J., Khan A.A., Hackney J.A., Lesch J., DeVoss J., Weirauch M.T., Singh H, & Mellman I. (2017). Transcriptional determinants of tolerogenic and immunogenic states during dendritic cell maturation. The Journal of Cell Biology, 216(3), 779-792.

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In Vitro Generation of Dendritic Cells

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DCs were generated in vitro from bone marrow cells. BM cells were cultured in complete RPMI containing 10% heat-inactivated FBS in the presence of 20 ng/ml rm GM-CSF (Peprotech) for 7 days. On day 4 fresh medium containing 20 ng/ml rmGM- CSF and 50 ng/ml rm IL-4 (Peprotech) was added. Nonadherent CD11c+ DCs were sorted using anti-CD11c-coated magnetic beads, according to the manufacturer’s directions (Miltenyi Biotec, Auburn, CA, USA), on Day 7. The absence of T cells in CD11c+ BMDCs preparations was confirmed by a Vβ8.2 -Jβ1.5 TCR specific PCR. To evaluate HEL responses in vitro BMDCs at responder:stimulator cell ratio 1 : 10 were co-cultured with or without sorted; CD4⁺CD25 ^med/lowCD62L^+/−CD44^high memory T cells in presence or absence of 10 μg /ml HEL. After 96 hr cells were harvested and stabilized with RNAlater (Ambion) for subsequent RNA isolation and library preparations.

Marusina A.I., Ono Y., Merleev A.A., Shimoda M., Ogawa H., Wang E.A., Kondo K., Olney L., Luxardi G., Miyamura Y., Yilma T.D., Villalobos I.B., Bergstrom J.W., Kronenberg D.G., Soulika A.M., Adamopoulos I.E, & Maverakis E. (2016). CD4+ Virtual Memory: Antigen-inexperienced T cells reside in the naïve, regulatory, and memory T cell compartments at similar frequencies, implications for autoimmunity. Journal of autoimmunity, 77, 76-88.

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Modulating Immune Response with IL-4

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Carrier-free rmIL-4 (1.5 µg/mouse, Peprotech) was mixed in PBS (200 µl/mouse) with neutralizing anti-mouse IL-4 antibodies (50 µg/mouse, Bio × Cell, clone 11B11, catalog number BE0045). Control mice received only an anti-trinitrophenol Rat IgG1 isotype control (50 µg/mouse, Bio × Cell, clone TNP6A7, catalog number BE0290). Mice were injected daily for 4 days intraperitoneally before harvesting thymi and spleens.

Istaces N., Splittgerber M., Lima Silva V., Nguyen M., Thomas S., Le A., Achouri Y., Calonne E., Defrance M., Fuks F., Goriely S, & Azouz A. (2019). EOMES interacts with RUNX3 and BRG1 to promote innate memory cell formation through epigenetic reprogramming. Nature Communications, 10, 3306.

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Stimulation of Mouse B Cells

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Mouse B cells (500,000) were cultured for 4 days in 2 mL R10 media and stimulated with 50 µg/mL LPS (MilliporeSigma), LPS with 10 ng/mL rmIL-4 (PeproTech), or LPS with 10 U/mL recombinant mouse IFN-γ (rmIFN-γ; PeproTech). B cells were analyzed by FACS on day 4. The culture supernatants were analyzed by ELISA for antibody secretion.

Chen H.Y., Almonte-Loya A., Lay F.Y., Hsu M., Johnson E., González-Avalos E., Yin J., Bruno R.S., Ma Q., Ghoneim H.E., Wozniak D.J., Harrison F.E, & Lio C.W. (2022). Epigenetic remodeling by vitamin C potentiates plasma cell differentiation. eLife, 11, e73754.

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