Recombinant il 3

Manufactured by BioLegend

Sourced in United States

Recombinant IL-3 is a cytokine that stimulates the growth and differentiation of hematopoietic progenitor cells. It is produced using recombinant DNA technology.

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Lab products found in correlation

Anti il 3 antibody, by BioLegend (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions) Easysep magnet, by STEMCELL (1 mentions) Hepes, by Thermo Fisher Scientific (1 mentions) β mercaptoethanol, by Merck Group (1 mentions) Amd3100, by Abcam (1 mentions) Naive t cell isolation kit, by Miltenyi Biotec (1 mentions) Mononylon 5, by Johnson & Johnson (1 mentions) Stereotactic frame, by Stoelting (1 mentions) Gm csf, by BioLegend (1 mentions) M csf, by BioLegend (1 mentions) Sodium pyruvate, by Merck Group (1 mentions)

6 protocols using recombinant il 3

Isolation and Culture of Mouse Mast Cells

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Mouse bone marrow-derived mast cells (BMMCs) were cultured as described (9 (link)). Mouse bone marrow-derived basophils were cultured in cRPMI supplemented with recombinant IL-3 at 20ng/ml (Biolegend, San Diego, CA), for 7-10 days, then sorted by flow cytometry selecting for CD49b-positive cells (Biolegend). Peritoneal lavage cells were cultured in cRPMI containing IL-3 (5ng/ml) and SCF (10ng/ml) for 5 days. Mast cells were positively selected using the EasySep Magnet from StemCell Technologies (Vancouver, BC), with c-Kit as a positive marker. Flow cytometry confirmed purity, which was essentially 100%.

Kolawole E.M., McLeod J.J., Ndaw V., Abebayehu D., Barnstein B.O., Faber T., Spence A.J., Taruselli M., Paranjape A., Haque T.T., Qayum A.A., Wijesinghe D.S., Sturgill J.L., Chalfant C.E., Straus D.B., Oskeritzian C.A, & Ryan J.J. (2016). Fluvastatin Suppresses Mast Cell and Basophil IgE Responses: Genotype-Dependent Effects. Journal of immunology (Baltimore, Md. : 1950), 196(4), 1461-1470.

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Isolation and Differentiation of Murine Bone Marrow Mast Cells

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Bone marrow mast cell (BMMC) isolation and differentiation was performed as previously described (32 (link)). Briefly, the femur and tibia of C57BL/6 mice were collected and the muscles were removed, and then the bone marrow (including the stem cells and progenitor cells) were flushed from the tibia and femur with repeated injections of ice cold PBS with a syringe using aseptic techniques. The harvested cells were then washed twice with PBS, and then resuspend and differentiated in culture media [RPMI 1,640 medium containing 2 mM L-glutamine, 15% FCS, 1 x Penicillin/streptomycin, 10 mM HEPES (all from Thermo Fisher, Hampton, NH, USA), 0.07% β-mercaptoethanol (Sigma, St. Louis, MO, USA), and 30 ng/mL recombinant IL-3 (BioLegend^®, San Diego, CA, USA)]. Fresh media was added every third day to the culture and the differentiation of BMMC was completed after 6 weeks of culture. Differentiation was monitored by FACS analysis (described under flow cytometer analysis).
After 6 weeks of differentiation, the isolated BMMCs were stimulated with mature recombinant mMCP6 prepared above and recombinant mMCP7 for specified times. BMMCs were lysed for RNA extraction and real-time quantitative reverse-transcriptase PCR.

Pattabiraman G., Liu Z., Paul M., Schaeffer A.J, & Thumbikat P. (2022). mMCP7, a Mouse Ortholog of δ Tryptase, Mediates Pelvic Tactile Allodynia in a Model of Chronic Pelvic Pain. Frontiers in Pain Research, 2, 805136.

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Modulating IL-3 Complexes and CXCR4 Inhibition

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For the treatment of IL-3 complexes, 10 μg of recombinant IL-3 (BioLegend) was mixed with 5 μg of anti-IL-3 antibody (BioLegend; clone: MP2-8F8, catalog#: 503910) to form an IL-3 complex^{53 (link)}. The IL-3 complex or control PBS was then intraperitoneally administered to mice. Single cell suspensions were prepared from the peripheral blood of mice 4 days after administration and subjected to flow cytometric analysis. For CXCR4 inhibitor treatment, mice were intraperitoneally treated with a single administration of AMD3100 (200 μg per mouse; abcam) or control PBS. Single cell suspensions were prepared from the spleen, and peripheral blood 1 h after treatment and subjected to flow-cytometric analysis.

Miyake K., Ito J., Nakabayashi J., Shichino S., Ishiwata K, & Karasuyama H. (2023). Single cell transcriptomics clarifies the basophil differentiation trajectory and identifies pre-basophils upstream of mature basophils. Nature Communications, 14, 2694.

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Microglia and T cell activation assay

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Sorted microglia were cultured in complete medium (RPMI-1640 supplemented with 10%FBS, 2mM L-glutamine, 10Uml⁻¹ penicillin and streptomycin, 10mM HEPES, 50μM 2-mercaptoethanol, 1mM sodium pyruvate and 1x nonessential amino acids) and kept in humidified 5% CO₂ incubator at 37°C. Microglia were exposed to 20ng/ml recombinant IL-3 (Biolegend) and/or 2μg/ml Beta-Amyloid (1-42) HiLyte™ conjugated to pHrodo iFL red (Invitrogen) for 3 hours. T-cells. Naive T cells were isolated from the spleen and lymph nodes using a Naive T cell isolation kit (Miltenyi Biotec) and cultured on anti-CD3 (2 μg/mL) coated plates in the presence of soluble anti-CD28 (2 μg/mL) and rmIL-2 (10 μg/mL) for 3 days and re-stimulated with PMA (100 ng/mL) and ionomycin (500 ng/mL) in the presence of GolgiPlug and GolgiStop (1:1000) for 3.5 hours prior to cell surface staining and analysis.

McAlpine C.S., Park J., Griciuc A., Kim E., Choi S.H., Iwamoto Y., Kiss M.G., Christie K.A., Vinegoni C., Poller W.C., Mindur J.E., Chan C.T., He S., Janssen H., Wong L.P., Downey J., Singh S., Anzai A., Kahles F., Jorfi M., Feruglio P.F., Sadreyev R.I., Weissleder R., Kleinstiver B.P., Nahrendorf M., Tanzi R.E, & Swirski F.K. (2021). Astrocyte-derived interleukin-3 reprograms microglia and limits Alzheimer’s disease. Nature, 595(7869), 701-706.

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Intracerebral Delivery of rIL-3 via Cannula and Osmotic Minipump

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Cannula and osmotic minipump (Alzet) implantation were performed as previously described^{11 (link)}. Briefly, mice were anesthetized, the head was shaved and secured in a stereotactic frame (Stoelting). An incision was made above the skull extending behind the shoulder blades. A small hole was drilled in the skull at AP −1; ML −0.27 from bregma and depth 2mm from dura to target the lateral ventricle. The cannula was inserted and glued to the skull. The cannula was connected to an osmotic minipump filled with recombinant IL-3 (Biolegend) conjugated to an anti-IL-3 antibody (Biolegend) as previously described³. Minipumps delivered rIL-3 into the ventricle at a rate of 1μ/day. Minipumps were implanted subcutaneously caudal the shoulder blades. At the end of the procedure the incision was sutured using mononylon 5.0 (Ethicon).

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Generation of Murine Bone Marrow-derived Cell Subsets

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Bone marrow was obtained from mouse femurs. The distal and proximal ends of the femurs were cut off and the bone marrow was flushed out with cold PBS using a syringe. Total cells were count (Neubauer chamber), washed once with sterile PBS. A total amount of 6 x 10 6 or 3 x 10 6 bone marrow cells were cultured in 10 ml RPMI complete medium (glutamine 20 µM, 10 % FCS, β-mercaptoethanol 50 µM, gentamycin 50 µg/ml) supplemented with 10 ng/ml M-CSF (Biolegend) for BMM generation or 10 ng/ml GM-CSF (Biolegend) for BMDC generation. At day 3, 5 ml BMM and 10 ml BMDC medium was added to the corresponding cells. At day 6, total BMM medium was replaced with 10 ml fresh one while half of the BMDC medium was replaced with fresh one. After 7 days, the cells were ready for experimental use.
For the generation of BMMCs, 10 x 10 6 bone marrow cells were cultured in RPMI 1640 medium containing + L-glutamine, 10 mM HEPES, 10% FCS, 50 µM gentamicin, 50 µM β-mercaptoethanol, 70 µM non-essential amino acids, 1 mM sodium pyruvate (Sigma) and recombinant IL-3 (10 ng/ml, Biolegend). BMMCs were transferred into a new petri dish with new BMMC medium every 3-4 days. BMMCs were ready to use after one month.

Er-Lukowiak M., Duan Y., Rassendren F., Ulmann L., Nicke A., Ufer F., Friese M.A., Koch‐Nolte F., Magnus T., & Rissiek B. (2020). A P2rx7 passenger mutation affects the vitality and function of immune cells in P2X4ko and other transgenic mice.

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