Mil 4

Manufactured by Thermo Fisher Scientific

Sourced in United States

The MIL-4 is a compact, high-performance laboratory centrifuge designed for general-purpose sample separation and processing. It features a brushless motor, programmable speed and time controls, and a durable, easy-to-clean metal housing. The MIL-4 is suitable for a wide range of applications in research and clinical laboratories.

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Lab products found in correlation

27 protocols using mil 4

Isolation and Culture of Murine Dendritic Cells

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Three mice from each group were sacrificed at 5 days after the second injection. Tumors as well as spleens were collected and filtered through a 70-μm cell strainer (Falcon; Corning). A single-cell suspension was centrifuged at 1,000 rpm for 5 min and lysed using red blood cell lysis buffer.²⁶ (link)
Spleen-derived DCs were isolated according to the references described. Cells were cultured in RPMI 1640 with 10% FCS, 20 ng/mL murine GM-CSF (PeproTech, Rocky Hill, NJ, USA), and 10 ng/mL mouse interleukin-4 (mIL-4; PeproTech, Rocky Hill, NJ, USA) for 6 days. Half of the medium was changed every 2 days, and sufficient amount of GM-CSF and mIL-4 were added. Suspension cells were collected and cultured for 2–3 days in RPMI 1640 medium with 20 ng/mL GM-CSF, 10 ng/mL mIL-4, and 15 ng/mL TNF-α before the assays.

Sun T., Luo Y., Wang M., Xie T, & Yan H. (2019). Recombinant Oncolytic Vaccinia Viruses Expressing Human β-Defensin 2 Enhance Anti-tumor Immunity. Molecular Therapy Oncolytics, 13, 49-57.

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Murine Splenocyte Activation Assay

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Splenocytes were stimulated with 100 U/ml of mIL-2, 10 µg/ml mIL-4, 1 µg/ml mIL-5 (all Peprotech) and 2 µg/ml F(ab′)2 fragments goat anti-mouse IgM (Jackson ImmunoREsearch), 2 µg/ml hamster anti-mouse CD40 mAb (3/23, BD), or 100 nM ODN74 5′–AAAAAAAAAAAAAACGTTAAAAAAAAAAA–3′ (Microsynth). Proliferation was assessed using the cell proliferation dye CPD eFluor 670 (eBioscience) and viability by 7-amino-actinomycin D (7AAD) or TO-PRO3 (Invitrogen) and Annexin-V exclusion and flow cytometric analysis.

Ottina E., Peperzak V., Schoeler K., Carrington E., Sgonc R., Pellegrini M., Preston S., Herold M.J., Strasser A, & Villunger A. (2017). DNA-binding of the Tet-transactivator curtails antigen-induced lymphocyte activation in mice. Nature Communications, 8, 1028.

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Isolation and Polarization of Mouse Macrophages

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Mouse femurs and tibias were excised from C57BL/6 mice (Charles River, UK) on a normal diet. The bone ends were cut and bone marrow was flushed out with sterile phosphate-buffered saline (PBS). Erythrocytes were removed using ACK lysis buffer (Life Technologies, UK) and the pelleted white blood cells resuspended in PBS and counted, giving an average yield of 40 × 10⁶ cells per mouse. Cells were plated into 24-well plates at 0.5 × 10⁶ cells/well and grown in RPMI 1640 media (Life Technologies) supplemented with antibiotics, glutamine, and 20% fetal calf serum (FCS) (Life Technologies) in the presence of 20 ng/ml recombinant human macrophage colony-stimulating factor (M-CSF, R&D Systems, USA). The media was changed every 3–4 days. After 7–10 days, when the cells had differentiated into macrophages, M-CSF was removed and the cells exposed to selected cytokines for 18 h in serum-free media (SFM), as specified in the text. These included mouse (m) IFNγ at 20 ng/ml (Miltenyi Biotec, USA), human (h) tissue necrosis factor (TNF)-α at 10 ng/ml (R&D Systems, USA), mIL-4 at 10 ng/ml (PreproTech, UK), and lipopolysaccharide (LPS) at 10 ng/ml (Sigma-Aldrich, USA, L2654). Human cytokines were used only when their efficacy on mouse cells had been previously documented by the suppliers.

Hayes E.M., Tsaousi A., Di Gregoli K., Jenkinson S.R., Bond A.R., Johnson J.L., Bevan L., Thomas A.C, & Newby A.C. (2014). Classical and Alternative Activation and Metalloproteinase Expression Occurs in Foam Cell Macrophages in Male and Female ApoE Null Mice in the Absence of T and B Lymphocytes. Frontiers in Immunology, 5, 537.

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Isolation and Activation of Immune Cells

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Primary dendritic cells, B cells, CD4⁺ T cells, and macrophages from C57BL/6J wild-type and miR-155 KO mice were cultured in RPMI medium with 10% FBS. Prior to harvesting primary dendritic cells, mice were subcutaneously injected with 1 × 10⁶ B16 melanoma cells expressing Flt3 ligand for about two weeks. After purification of splenic CD11c⁺ dendritic cells by CD11c microbeads (Miltenyi Biotec), dendritic cells were activated in a medium containing 100 ng/ml LPS (SIGMA) and 20 ng/ml GM-CSF (Tonbo). Splenic primary B cells were purified by negative selection using Dynabeads Mouse CD43 (Invitrogen), and activated in a medium containing 25 μg/ml LPS and 6.5 ng/ml mIL-4 (PeproTech). CD4⁺ T cells from lymph node and spleen were purified with Dynabeads FlowComp Kit (Invitrogen). CD4⁺CD25⁻CD44⁻ T cells were then activated with Dynabeads Mouse T-Activator CD3/CD28 (Invitrogen). Intraperitoneal macrophages, induced by thioglycollate injection, were harvested and activated with 100 ng/ml LPS.

Hsin J.P., Lu Y., Loeb G.B., Leslie C.S, & Rudensky A.Y. (2018). The effect of cellular context on miR-155-mediated gene regulation in four major immune cell types. Nature immunology, 19(10), 1137-1145.

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Murine CD4+ T Cell Isolation and Functional Assay

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Fetal bovine serum (FBS), RPMI1640, and fluorescence dyes Dio and Dil were purchased from Life Technologies (California, USA). Recombinant mIL-3 and mIL-4 were purchased from PeproTech (Rocky Hill, NJ, USA). CD4⁺CD62L⁺ T cell Isolation Kit II was purchased from Miltenyi Biotec (Paris, France). FITC-labeled rat anti-mouse mAbs directed against CD117, PE-labeled rat anti-mouse mAbs directed against FcεRI, FITC-labeled rat anti-mouse mAbs directed against CD4, PE-labeled rat anti-mouse mAbs directed against IL-4, and PerCP/Cγ5.5-labeled rat anti-mouse mAbs directed against IFN-γ were purchased from Biolegend (San Diego, CA). Goat anti-mouse OX40 mAb and rat anti-mouse OX40L mAb were obtained from R&D System (Minneapolis, MN, USA). Cell Counting Kit -8 (CCK-8, DojinDo, Japan) was used to assess the proliferation rate of cells. Antimast cell tryptase antibody was purchased from Abcam (America). Anti-rat IgG-HRP was purchased from Dako (Japan). ECL+ system was purchased from Amersham (Piscataway, NJ). All the information of primary antibodies is included in Table 1.

Li F., Wang Y., Lin L., Wang J., Xiao H., Li J., Peng X., Dai H, & Li L. (2016). Mast Cell-Derived Exosomes Promote Th2 Cell Differentiation via OX40L-OX40 Ligation. Journal of Immunology Research, 2016, 3623898.

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Murine Dendritic Cell Culture Protocol

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A20 B cell lymphoma cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained via ATCC protocol.
Dendritic cells preparation was performed as previously described and modified [4 (link)]. Bone marrow–derived murine dendritic cells were generated by culturing bone marrow cells from the femur and tibiae of BALB/c mice at a starting concentration of 1×10⁶ cells/mL in RPMI-1640/10% fetal bovine serum (FBS; Invitrogen, Carlsbad, California) supplemented with 30 ng/mL of recombinant murine granulocyte macrophage colony-stimulating factor (mGM-CSF; PeproTech, Rocky Hill, NJ) and 3 ng/mL interleukin 4 (mIL-4; PeproTech, Rocky Hill, NJ). Fresh medium supplemented with GM-CSF plus IL-4 was added on day 3, and all of the loosely adherent cells were transferred to Petri dishes on day 6. 2 days later, nonadherent cells and loosely adherent dendritic cells were harvested, washed with PBS.

Zhu X.J., Yang Z.F., Zhou J.Y., Liu L., Sun X.M., Fan Z.F., Hu S.Y., Chen Y.C., Li W.X., Cao M, & Wang L.X. (2015). Progression of Large Lymphoma Is Significantly Impeded with a Combination of Gemcitabine Chemotherapy and Dendritic Cells Intra-Tumor Vaccination. PLoS ONE, 10(7), e0132799.

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Isolation and Activation of Immune Cells

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Isolation and Activation of Murine BMDMs

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Murine bone marrow-derived macrophages (BMDM) were isolated and characterized as previously described(22 (link)). Briefly, bone marrow was harvested from 6- to 8-week-old B6 mice, B6 st2^−/− mice, or Balb/c st2^−/− mice. Harvested cells from the bone marrow were washed and plated at 2 × 10⁶ cells/mL and were allowed to differentiate into macrophages for 7 days in the presence of macrophage colony-stimulating factor (MCSF) with complete medium changes every 48 h. Macrophages were then activated for 24 h with one of the following: 1) 20 ng/mL Interferon-γ (IFNγ) and 100 ng/mL lipopolysaccharide (LPS) (Affymetrix eBioscience, Santa Clara, CA; Sigma Aldrich) to promote an M_IFNγ+LPS phenotype (M1-like); 2) 20 ng/mL interleukin (IL)-4 (Invitrogen) to promote an M_IL-4 phenotype (M2-like); 3) 20 ng/ml IL-33 (Peprotech), or 4) 25 µg/mL of wt mouse MBV, IL-33^−/− mouse MBV, or porcine SIS-MBV. After the incubation period at 37°C, cells were washed with sterile PBS and lysates prepared using RIPA buffer for immunoblot analysis, or cells fixed with 2% paraformaldehyde (PFA) for immunolabeling.

Hussey G.S., Dziki J.L., Lee Y.C., Bartolacci J.G., Behun M., Turnquist H.R, & Badylak S.F. (2019). Matrix bound nanovesicle-associated IL-33 activates a pro-remodeling macrophage phenotype via a non-canonical, ST2-independent pathway. Journal of immunology and regenerative medicine, 3, 26-35.

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Generating Murine Dendritic Cells

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Mouse bone marrow cells were isolated from 8‐week‐old C57BL/6J male mice (Beijing HFK Bioscience, Beijing, China) and cultured for 7 days in RPMI‐1640 medium (Gibco) containing 10% FBS (Biological Industries), supplemented with 20 ng·mL^–1 murine granulocyte macrophage‐colony stimulating factor (mGM‐CSF; PeproTech), and 20 ng·mL^–1 mIL‐4 (PeproTech) at 37 °C in a 5% CO₂ incubator. The generated DCs were immature and their purity was determined by flow cytometry analysis of CD11c⁺ cells. Animal experiments were approved by the Animal Ethical and Welfare Committee of Shandong University (AEWC number: 18021) and were compliant with the Guide for the Care and Use of Laboratory Animals. Mice were housed in a rectangular mouse cage (area: 635 cm², height: 18 cm) and were kept in a specific pathogen‐free environment under standard experimental conditions (light–dark cycle: 12 h, temperature: 20–22 °C, humidity: 50–70%) with ad libitum access to food and water. Five mice were housed in one cage and were cared for every day.

Li Y., Song Z., Han Q., Zhao H., Pan Z., Lei Z, & Zhang J. (2022). Targeted inhibition of STAT3 induces immunogenic cell death of hepatocellular carcinoma cells via glycolysis. Molecular Oncology, 16(15), 2861-2880.

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Cytokine-driven hematopoietic cell culture

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Bone marrow cells were obtained from Wild-type (WT) BALB/c, IL-9-GFP (20 (link)) and Batf^−/− (21 , 22 ) mice as previously described (24 (link)). Briefly, cells were cultured in complete media containing mSCF and mIL-3 (both 20 ng/ml) (Peprotech, Inc. Rocky Hill, New Jersey) for the first 6 days. On day 3, non-adherent cells were collected and resuspended in media containing mSCF and mIL-3. On day 6, non-adherent cells were obtained, spun down and suspended in media containing either SCF and IL-3 or the combination of mSCF, mIL-3 and mIL-4 (20ng/ml) (Peprotech, Inc. Rocky Hill, New Jersey). Cells were cultured for 4 more days, non-adherent cells were collected and suspended in media with fresh cytokines on d10 and resuspended at 1× 10⁶ cells/ml, 200μl/well in 96 well plates. Cells were stimulated with mIL-33 (100ng/ml) for 24 hours.

Tomar S., Ganesan V., Sharma A., Zeng C., Waggoner L., Smith A., Kim C.H., Licona-Limón P., Reinhardt R.L., Flavell R.A., Wang Y.H, & Hogan S.P. (2020). IL-4-BATF signaling directly modulates IL-9 producing mucosal mast cell (MMC9) function in experimental food allergy. The Journal of allergy and clinical immunology, 147(1), 280-295.

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