Recombinant mouse il 3

Manufactured by BioLegend

Recombinant mouse IL-3 is a cytokine that supports the growth and differentiation of hematopoietic progenitor cells. It is produced in a mammalian expression system.

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8 protocols using recombinant mouse il 3

Synthesis and Use of Didox Compound

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3,4-Dihydroxybenzohydroxamic acid (Didox) was synthesized by Molecules for Health, Inc. (Richmond, VA). Lyophilized Didox was resuspended in DEPC-treated water at concentrations of 100mM, briefly sonicated, and filter sterilized (0.45mm syringe filter, Cell Treat). Didox was added to cultures at a final concentration of 100µM unless otherwise indicated. Recombinant mouse IL-3 and SCF were purchased from Biolegend (San Diego, CA). DNP-specific purified mouse IgE was purchased from BD Pharmingen (San Jose, CA). Dinitophenyl-coupled human serum albumin (DNP-HSA), propidium iodide, N-acetylcysteine (NAC), and hydroxyurea (HU) were purchased from (Sigma, St Louis, MO).

McLeod J.J., Caslin H.L., Spence A.J., Kolawole E.M., Abdul Qayum A., Paranjape A., Taruselli M., Haque T.T., Kiwanuka K.N., Elford H.L, & Ryan J.J. (2017). Didox (3,4-dihydroxybenzohydroxamic acid) suppresses IgE-mediated mast cell activation through attenuation of NFκB and AP-1 transcription. Cellular immunology, 322, 41-48.

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In Vitro Differentiation of Mouse Bone Marrow-Derived Mast Cells

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Mouse BMMCs were differentiated in vitro from isolated WT or HDC−/− mice tibia and femur bone marrow cells in RPMI 1640 medium (Invitrogen) supplemented with 1 mM sodium pyruvate (Gibco), 1 mM HEPES (Invitrogen), 2 mM L-glutamine, 1% penicillin-streptomycin (Gibco), and 10% fetal calf serum (FCS, Gibco), with the presence of 1 ng/ml recombinant mouse IL-3 (Biolegend) and 10 ng/ml mouse SCF (Biolegend). The percentage of MCs was determined by flow cytometry after 4 weeks of differentiation. Purity >95% FCεRIα+c-kit+ BMMCs were subjected to further experiments. In some experiments, BMMCs were co-cultured with HDC−/− splenocytes in the presence of 10 ng/ml GM-CSF (Biolegend); 5 × 10⁻⁶ M histamine and/or 5 μg/ml OVA_323–339 peptide was added respectively for additional 96 h culture.

Chen X., Churchill M.J., Nagar K.K., Tailor Y.H., Chu T., Rush B.S., Jiang Z., Wang E.B., Renz B.W., Wang H., Fung M.C., Worthley D.L., Mukherjee S, & Wang T.C. (2015). IL-17 producing mast cells promote the expansion of myeloid-derived suppressor cells in a mouse allergy model of colorectal cancer. Oncotarget, 6(32), 32966-32979.

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Murine Bone Marrow-Derived Mast Cell Assay

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mBMMCs were generated by flushing BM precursors from tibias and femurs of C57BL/6 mice and culturing in RPMI supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 ng/ml recombinant mouse IL-3 (BioLegend), and 10 ng/ml mouse stem cell factor (PeproTech) for 4–6 wk at 37°C in 5% CO₂. HeLa cells that express human FcεRI- α, -β, and -γ chains (hFcεRI⁺-HeLa cells) were generated by retroviral transduction. BMMCs or hFcεRI⁺-HeLa cells were incubated overnight with 100 ng of mouse or human IgE, respectively. Cells were washed to remove unbound IgE before incubation with A647-OVA (500 ng/ml; Molecular Probes) for OVA-specific IgE or Alexa Fluor 647–streptavidin (1:200 dilution; BioLegend) for biotinylated hIgE for 10 min at 37°C. FcεRI expression of the IgE-sensitized cells was confirmed by staining with PE-labeled anti-hFcεRIα (CRA1-PE clone AER-37; eBioscience) and anti-mFcεRIα (MAR1-PE; BioLegend). Flow cytometric analysis was performed using the FACSCanto II and FACSDiva software (BD).

Shade K.T., Platzer B., Washburn N., Mani V., Bartsch Y.C., Conroy M., Pagan J.D., Bosques C., Mempel T.R., Fiebiger E, & Anthony R.M. (2015). A single glycan on IgE is indispensable for initiation of anaphylaxis. The Journal of Experimental Medicine, 212(4), 457-467.

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Hematopoietic Stem Cell Culture Conditions

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For growth assays, sorted CD34^-CD150⁺LSK HSCs were cultured in S-Clone SF-O3 (Sanko Junyaku) supplemented with 0.1% BSA (093001; STEMCELL Technologies), 50 μM 2-ME (Sigma-Aldrich) and 1% penicillin/streptomycin/glutamine (Gibco). 20 ng/mL of recombinant mouse SCF (579706; BioLegend) and recombinant human TPO (763706; BioLegend) for HSC culture conditions and 10 ng/mL of SCF, TPO, recombinant mouse IL-3 (575506; BioLegend), and recombinant murine GM-CSF (315-03; PeproTech) for myeloid culture condition-1 were added to cultures. In the case of myeloid culture condition-2, sorted CD150⁺CD48^-CD135^-CD34^-LSK HSCs, LSK cells, and GMPs were cultured in IMDM (Gibco) supplemented with 5% FBS, 50 μM 2-ME (Sigma-Aldrich), 1% penicillin/streptomycin/glutamine (Gibco), 1 mM sodium pyruvate (Gibco), and 0.1 mM MEM Non-Essential Amino Acids solution (Gibco). 25 ng/mL of SCF, TPO, recombinant human Flt3L (300-19; PeproTech) and recombinant murine IL-11 (220-11; PeproTech) and 10 ng/mL of IL-3 and GM-CSF were added to cultures.
For replating assays, LSK cells were plated in methylcellulose medium (Methocult M3234; STEMCELL Technologies) containing 20 ng/mL of SCF, TPO, IL-3, and GM-CSF.

Nakajima-Takagi Y., Oshima M., Takano J., Koide S., Itokawa N., Uemura S., Yamashita M., Andoh S., Aoyama K., Isshiki Y., Shinoda D., Saraya A., Arai F., Yamaguchi K., Furukawa Y., Koseki H., Ikawa T, & Iwama A. (2023). Polycomb repressive complex 1.1 coordinates homeostatic and emergency myelopoiesis. eLife, 12, e83004.

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Bone Marrow Derived Mast Cell Differentiation

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Bone marrow derived mast cells (BMMCs) were derived from hematopoietic progenitor cells from femurs of C57BL/6J mice (Jackson Laboratories). Cells were pooled from femurs of two mice into one T75 cell culture flask and cultured for 4–6 weeks at 37°C and 5% CO₂ in supplemented media containing IL-3 for mast cell differentiation. RPMI 1640 media (Corning Inc., Corning, NY) was supplemented with the following ingredients: 10% heat inactivated fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES buffer, MEM nonessential amino acids (Corning Inc., Corning, NY), 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL primomycin (Invitrogen, San Diego, CA), 0.000325% 2-mercaptoethanol, and 30 ng/mL recombinant mouse IL-3 (BioLegends, San Diego, CA). Maturity of BMMCs was confirmed between 4–6 weeks by measuring levels of surface receptors c-kit and FcεR1 via flow cytometry (BD Accuri, San Jose, CA) (Supplemental Figure 1A). Each biological replicate consists of a single flask of cells isolated from the femurs of two separate mice and combined. Therefore, each statistical N is a flask containing cells from two mice.

Mendoza R.P., Anderson C.C., Fudge D.H., Roede J.R, & Brown J.M. (2021). Metabolic consequences of IgE and non-IgE mediated mast cell degranulation. Journal of immunology (Baltimore, Md. : 1950), 207(11), 2637-2648.

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IL-3, SCF, and IL-33 Modulation

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Didox was supplied by Molecules for Health, Inc, (Richmond, VA). Recombinant mouse IL-3, SCF, and IL-33 were purchased from Biolegend (San Diego, CA). Hydroxyurea (HU), N-acetylcysteine (NAC), and lipopolysaccharide (LPS) from Escherichia coli 055:B5 (catalog L4524) were purchased from Sigma (St Louis, MO).

Caslin H.L., McLeod J.J., Spence A.J., Qayum A.A., Kolawole E.M., Taruselli M.T., Paranjape A., Elford H.L, & Ryan J.J. (2017). Didox (3,4-dihydroxybenzohydroxamic acid) suppresses IL-33-induced cytokine production in primary mouse mast cells. Cellular immunology, 319, 10-16.

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Recombinant Cytokines for Mast Cell Study

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Recombinant mouse IL-3, SCF and IL-10 were purchased from Biolegend (San Diego, CA). Purified mouse IgE (clone C38-2, κ isotype) was purchased from BD Biosciences (Pharmingen division, San Diego, CA). Dinitrophenyl-coupled human serum albumin (DNP-HSA) and LPS (Cat# L-6529) were purchased from Sigma-Aldrich (St. Louis, MO). Histamine was purchased from Enzo Life Sciences (Farmingdale, NY). All western blotting antibodies were purchased from Cell Signaling Technology (Danvers, MA). Dr. Daniel Conrad (VCU) generously provided purified mouse anti-DNP IgE for in vivo experiments. Antibodies for c-Kit (PE anti-mouse CD117) and FcεRI (APC anti-mouse FcεRIα) were purchased from Biolegend (San Diego, CA) and used at a concentration 1:100. LPS levels were tested using Toxin Sensor^™ Chromogenic LAL Endotoxin Assay Kit from GenScript (Piscataway, NJ). IL-10 resuspended in PBS and PBS alone used in in vivo studies had LPS content of <0.1 EU/mol. Media and IL-10 resuspended in media for in vitro studies had LPS content of ~1.0 EU/mol. There was no significant difference between IL-10 and the respective vehicle control used when comparing LPS levels using unpaired Student’s t-test.

Qayum A.A., Paranjape A., Abebayehu D., Kolawole E.M., Haque T.T., McLeod J.J., Spence A.J., Caslin H.L., Taruselli M.T., Chumanevich A.P., Baker B., Oskeritzian C.A, & Ryan J.J. (2016). IL-10-induced miR-155 targets SOCS1 to enhance IgE-mediated mast cell function. Journal of immunology (Baltimore, Md. : 1950), 196(11), 4457-4467.

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Identification of Immune Cell Populations

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The following Abs were purchased from BD Pharmingen: allophycocyanin (APC)-conjugated anti-CD49b (HM2), phycoerythrin (PE)-conjugated anti-Siglec-F (E50-2440), and anti-NK1.1 (PK136). Unlabeled anti-IL-3 (MP2-8F8) and rat IgG1 isotype (RTK2071); biotin-conjugated anti-CD49b (DX5), anti-CD123 (IL-3R, 5B11), anti-FcRI (MAR-1), anti-I-A/I-E (M5/114.15.2), American hamster IgG isotype (HTK888), and rat IgG2a isotype (RTK2758); PE-conjugated CD200R3 (Ba13), anti-IgE (RME-1), and anti-CD4 (RM4.5); APC-conjugated anti-Gr-1 (RB6-8C5), anti-B220 (RA3-6B2), and anti-F4/80 (BM8); PE/Cy7-conjugated anti-c-kit (2B8) and anti-CD45 (30-F11); Brilliant Violet 421-conjugated anti-CD3 (145-2C11) and anti-CD11c (N418); and APC/Cy7-conjugated streptavidin were from BioLegend. PE-conjugated CD11b Abs (M1/70) were obtained from eBioscience. Anti-STIM1 (D88E10), anti-STIM2 (4917), and anti--tubulin (2144) were from Cell Signaling Technology. TNP-specific IgE (IGEL-b4) and anti-CD16/32 (2.4G2) were prepared in our laboratory. TAM (T5648), DT (D0564), inactive mDT (D2189), EGTA, BAPTA-AM, and DPI were purchased from Sigma-Aldrich. Recombinant mouse IL-3 and IL-33 were obtained from BioLegend.

Yoshikawa S., Oh-Hora M., Hashimoto R., Nagao T., Peters L., Egawa M., Ohta T., Miyake K., Adachi T., Kawano Y., Yamanishi Y, & Karasuyama H. (2019). Pivotal role of STIM2, but not STIM1, in IL-4 production by IL-3-stimulated murine basophils. Science signaling, 12(576).

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