Interleukin 3 (il 3)

Manufactured by BD

Sourced in United States

IL-3 is a laboratory product used in cell culture and research applications. It is a recombinant human interleukin-3 protein that can be used to stimulate the growth and differentiation of various hematopoietic cell types. The core function of IL-3 is to support the proliferation and survival of blood cell precursors.

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8 protocols using interleukin 3 (il 3)

Isolation and Differentiation of Eosinophil and Mast Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated through Ficoll-Hypaque (Sigma) density centrifugation from blood obtained from healthy volunteers. CD34⁺ cells were enriched from PBMCs using positive magnetic affinity column purification (Miltenyi Biotec) and eosinophil progenitors were derived using the technique of Hudson et al. (22 (link)) by culturing purified CD34⁺ cells in complete medium (RPMI1640 and 10% FBS) supplemented with stem cell factor (SCF; 25 ng/ml; BD Biosciences), thymopoietin (TPO; 25 ng/ml; R&D System, Minneapolis, MN), Fms-like tyrosine kinase 3 (Flt3) ligand (25 ng/ml; BD Biosciences), IL-3 (25 ng/ml; BD Biosciences) and IL-5 (25 ng/ml; BD Biosciences) with or without IFN-γ (20 ng/ml; BD Bioscience) for 3 days and then cultured for an additional 3 weeks with just IL-3 and IL-5 (±IFN-γ). Cells were washed and fresh media and cytokines were applied weekly. Maturation was accessed as previously described (17 (link)) and cells were activated as described above.
Mast Cells. For studies involving mast cells, CD34⁺ cells isolated as described were cultured for 8 weeks with stem cell factor and IL-6 and, for the first week only, with IL-3 according to the methodology of Kirshenbaum et al (23 (link), 24 (link)).

Steinke J.W., Negri J., Liu L., Payne S.C, & Borish L. (2014). Aspirin Activation of Eosinophils and Mast Cells: Implications in the Pathogenesis of Aspirin-Exacerbated Respiratory Disease. Journal of immunology (Baltimore, Md. : 1950), 193(1), 41-47.

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Eosinophil Purification and Activation

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Circulating eosinophils were purified from donors with eosinophils >35 per μL of blood. The participants did not use systemic steroids or take topical/inhaled corticosteroids the day of the blood draw. Informed consent was obtained from each subject prior to participation. The studies were approved by the University of Wisconsin-Madison Health Sciences Institutional Review Board (#2013-1570). As previously described [17 (link)], eosinophils were purified by negative selection. Eosinophil preparations with purity >98% and viability ~98% were used. Eosinophils were cultured at 1 × 10⁶/mL in complete medium (RPMI 1640 plus 10% fetal bovine serum) with IL3 (2 ng/mL) or IL5 (2 ng/mL) (both cytokines from BD Biosciences [San Jose, CA, USA]) for 20 h, and were then washed and cultured with complete medium (no IL3 or IL5) on coated heat-aggregated immunoglobulins-G (HA-IgG or IgG) or without IgG as previously described [17 (link)]. Human serum IgG was from Sigma-Aldrich (St. Louis, MO, USA).

Esnault S., Fichtinger P.S., Barretto K.T., Fogerty F.J., Bernau K., Mosher D.F., Mathur S.K., Sandbo N, & Jarjour N.N. (2022). Autophagy Protects against Eosinophil Cytolysis and Release of DNA. Cells, 11(11), 1821.

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Eosinophil/Basophil Progenitor Assay

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Enriched CD34+ progenitors (8000 cells/well) were cultured in duplicates in 0.9% methylcellulose (Sigma Aldrich, St. Louis, MO, USA) with Iscove's 2+ (modified Dulbecco's medium (Gibco, Burlington, Ontario, Canada) supplemented with FBS, penicillin–streptomycin, and 2-ME) and IL-3 (1 ng/mL), IL-5 (1 ng/mL), or GM-CSF (10 ng/mL; BD Biosciences, Mississauga, ON, Canada) in the presence or absence of TSLP (10 ng/mL; PeproTech, Rocky Hill, NJ, USA) in 12-well plates (Corning Costar, Corning, NY, USA). In some experiments, cells were treated with anti-TSLP (Amgen, Seattle, WA, USA), anti-TSLPR (R&D Systems, Minneapolis, MN, USA), anti-TNFα (R&D), or isotype control (each at 10 µg/mL). Treatment with the indicated stimulatory/inhibitory conditions had no effects on cell viability as determined by trypan blue exclusion. Cultures were incubated for 14 days (37°C, 5% CO₂). Eo/B CFU were enumerated using inverted light microscopy (colonies were defined as tight, granular clusters ≥40 cells).

Hui C.C., Rusta-Sallehy S., Asher I., Heroux D, & Denburg J.A. (2014). The effects of thymic stromal lymphopoietin and IL-3 on human eosinophil–basophil lineage commitment: Relevance to atopic sensitization. Immunity, Inflammation and Disease, 2(1), 44-55.

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Angiogenic Potential of HUVEC-Pericyte Co-culture

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Lentivirus green fluorescent protein transduced HUVECs (HUVECs-GFP) and lentivirus discosoma sp. Red fluorescent protein transduced pericytes (pericytes-DsRED) were mixed at 5:1 ratio in co-culture medium, which is basal EBM medium supplied with 2% FCS, rhFGF-B, ascorbic acid and 100 UmL^-1 PS. Cell mixture supplied with growth factors, including IL-3, SCF-1 and CXCL12 (BD Bioscience), at the volume of 300 µL was added to 200 µL bovine collagen type I (Gibco). NaOH was used to adjust pH to 7.5. Cell-collagen mixture was added to 96-well plate (50 µL per well). After 1 h incubation, 100 µL EGM2 was added per well and the plate was incubated overnight. The following day IS or control buffer was added to the cells. Images were taken from day 1 till day 3 after stimulation using inverted fluorescence microscope and analysed by AngioSys 2.0. To correct for batch effects, an additional condition of cells cultured in the standard co-culture medium was included in each independent experiment.

Pei J., Juni R., Harakalova M., Duncker D.J., Asselbergs F.W., Koolwijk P., van Hinsbergh V., Verhaar M.C., Mokry M, & Cheng C. (2019). Indoxyl Sulfate Stimulates Angiogenesis by Regulating Reactive Oxygen Species Production via CYP1B1. Toxins, 11(8), 454.

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Multiplex Cytokine Profiling in Serum

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Cytometric bead array (CBA) kits were used to detect 14 cytokines/chemokines, among which 7 analytes including IL-5 (BD#558278), IL-3 (BD#558355), IL-7 (BD#558334), IL-8 (BD#558277), IL-13 (BD#558450), MCP1 (BD#558287) and IP10 (BD#558280) were detected using ordinary BD CBA kits, 4 analytes including IL-1β (BD#561509), IL-4 (BD#561510), TNF (BD#561516) and IFN-γ (BD#561515) were detected using enhanced sensitivity (ES) BD CBA kits, 3 cytokines including IL-2, IL-6 and IL-10 were detected using human Th1/Th2 assay kit purchased from Cell-gene Bio-Engineering Co., Ltd (Hangzhou, China). Serum IL-16 was tested by ELISA (R&D#D1600). All cytokine/chemokine detection was performed according to the manufacturer’s instructions.

Jia R., Wang X., Liu P., Liang X., Ge Y., Tian H., Chang H., Zhou H., Zeng M, & Xu J. (2020). Mild Cytokine Elevation, Moderate CD4+ T Cell Response and Abundant Antibody Production in Children with COVID-19. Virologica Sinica, 35(6), 734-743.

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Epithelial-Mesenchymal Transition Markers in Breast Cancer Cell Lines

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Real-time polymerase chain reaction (PCR) was performed to detect SLUG, ZEB 1, and TWIST transcript in Hs-578T, HCC-1395, MDA-MB-231, and MDA-MB-436 cell lines untreated or treated with IL-3 (5 ng/mL of recombinant human IL-3) (BD Biosciences, San Jose, CA, USA) for 24 h. The Primer sequences and additional details are reported in Supplementary Information.

Koni M., Castellano I., Venturelli E., Sarcinella A., Lopatina T., Grange C., Cedrino M., Femminò S., Cossu-Rocca P., Orrù S., D’Ascenzo F., Cotellessa I., Tampieri C., Debernardi C., Cugliari G., Matullo G., Camussi G., De Miglio M.R, & Brizzi M.F. (2022). Interleukin-3-Receptor-α in Triple-Negative Breast Cancer (TNBC): An Additional Novel Biomarker of TNBC Aggressiveness and a Therapeutic Target. Cancers, 14(16), 3918.

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Cell Culture Protocols for Cell Lines

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Phoenix GP, 293TN and NIH 3T3 cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), 80 U/ml penicillin, and 80 mg/ml streptomycin. FDC-P1 cell line was cultured in DMEM supplemented with 25% mouse interleukin-3 (IL-3, BD Biosciences, San Jose, CA, USA) and 10% FBS, 80 U/ml penicillin and 80 mg/ml streptomycin. These cell lines were incubated in a humidified atmosphere of 5% CO₂ in air at 37 °C. All cell lines used in this study were purchased from American Type Culture Collection (ATCC).

You Y., Wen R., Pathak R., Li A., Li W., St Clair D., Hauer-Jensen M., Zhou D, & Liang Y. (2014). Latexin sensitizes leukemogenic cells to gamma-irradiation-induced cell-cycle arrest and cell death through Rps3 pathway. Cell Death & Disease, 5(10), e1493-.

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Retroviral transduction of FL5.12 pro-B cells

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The FL5.12 pro-B cell line [41] (link) was a gift from Dr. Craig Thompson (Memorial Sloan-Kettering Cancer Center). FL5.12 cells were cultured in RPMI 1640 media with 10% FBS (Thermo Scientific), 10 mM Hepes pH 7.4, 100 U/ml Penicillin, 100 mg/ml Streptomycin, 55 mM β-Mercaptoethanol (all from Gibco), supplemented with 0.6 ng/ml IL3 (BD Pharmingen). To prepare retroviral supernatant for infection, 293T cells at ∼70% confluency were transfected with Effectene reagent (Qiagen) according to manufacturer’s instructions. The pSiren (Clontech) library was co-transfected with an ecotropic retroviral packaging plasmid pCL-Eco (Imgenex) at a dose of 2.5 µg total DNA per well in a 6-well plate. Supernatant was harvested to infect FL5.12 cells with 3 cycles of centrifugation (2500 g for 45 minutes) and incubation (2 hrs), in the presence of 5 µg/ml polybrene (Sigma). Infection efficiency was monitored by mCherry expression on a BD LSRII flow cytometer. Ideally the mCherry percent positivity was kept at ∼33% or less whenever a library was used to transduce cells, so that, by Poisson distribution, the majority of the infected cells received only one construct.

Wang Y., Speier J.S., Engram-Pearl J, & Wilson R.B. (2014). Introduction of Mismatches in a Random shRNA-Encoding Library Improves Potency for Phenotypic Selection. PLoS ONE, 9(2), e87390.

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