Mouse il 3

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom, United States

Mouse IL-3 is a recombinant mouse interleukin-3 protein. Interleukin-3 is a hematopoietic growth factor that stimulates the proliferation and differentiation of various blood cell progenitors.

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23 protocols using mouse il 3

Culturing Bone Marrow Cells from Ataxin-3 Mice

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Bone marrow cells were collected from femurs of homozygous (304Q/304Q) and heterozygous (WT/304Q) 12 month old 304Q ataxin-3 knock-in and wild type (WT/WT) mice and cultured in RPMI (Gibco, Thermo Fisher Scientific, Waltham, MA), supplemented with 10% fetal bovine serum, penicillin/streptomycin (Biochrom, Berlin, Germany), plus 30 ng/mL mouse IL-3 (PeproTech, Hamburg, Germany). Cells were cultured in 6 well plates, incubated at 37°C in a humidified incubator under 5% (v/v) CO2. Starting after 3 days, half of the medium was changed once a week every week. In addition, starting after 3 weeks, medium was changed completely once a week every week by transferring the non-adherent cells in fresh medium into a new plate. After culture for 9 weeks, cells were used for functional assays. For each genotype, several lines were generated. Maturity and purity of the BMMC were examined on cytospins stained with May-Grünwald/Giemsa (Carl Roth). We did not detect differences in the staining of pure BMMC from 304Q/304Q mice compared to BMMC from WT/WT mice (not shown).

Sowa A.S., Haas E., Hübener-Schmid J, & Lorentz A. (2022). Ataxin-3, The Spinocerebellar Ataxia Type 3 Neurodegenerative Disorder Protein, Affects Mast Cell Functions. Frontiers in Immunology, 13, 870966.

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Hematopoietic Stem Cell Culture Protocol

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Cells were clone sorted into 96-well round-bottom plates. HSCs were cultured in S-Clone (Iwai North America Inc.) supplemented with 0.75% AlbuMAX-I (Gibco), 1x penicillin/streptomycin, 50 mM 2-mercaptoethanol (Invitrogen) and the following cytokines: 20 ng/ml mouse stem cell factor, 20 ng/ml mouse thrombopoietin, 20 ng/ml mouse IL-12. Myeloid progenitors and c-kit enriched cells were cultured in Dulbecco’s modified Eagle’s medium and F-12 medium (Gibco and Invitrogen) supplemented with 10% fetal calf serum (Hyclone and Thermo Scientific), 1x penicillin/streptomycin, 2 mM GlutaMAX, 50 mM 2-mercaptoethanol (Invitrogen), and the following cytokines: 20 ng/ml mouse stem cell factor, 20 ng/ml mouse thrombopoietin, 20 ng/ml mouse IL-3, 20 ng/ml mouse granulocyte macrophage colony-stimulating factor (all purchased from PeproTech). c-kit enriched bone marrow (2×10⁶ cells/ml) were exposed for 4 hours to 10 μM ATMi (KU55933, Selleckchem). All cells were kept in a 5% O₂ incubator.

Gutierrez-Martinez P., Hogdal L., Nagai M., Kruta M., Singh R., Sarosiek K., Nussenzweig A., Beerman I., Letai A, & Rossi D.J. (2018). Diminished apoptotic priming and ATM signalling confer a survival advantage onto aged haematopoietic stem cells in response to DNA damage. Nature cell biology, 20(4), 413-421.

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Generating Leukemic Npm1c/Flt3-ITD/Cas9 Cells

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Npm1c/Flt3-ITD/Cas9 DM cells were generated from lineage-depleted bone marrow cells of primary transgenic mice after leukemia onset (female, 12 weeks old) as described previously^{57 (link),58 (link)}. Cells were maintained in XVIVO-20 medium (Lonza) supplemented with 5% FCS, 1% penicillin-streptomycin-glutamine, mouse SCF 50 ng ml⁻¹ (PeproTech), mouse IL-3 10 ng ml⁻¹ (PeproTech) and mouse IL-6 10 ng ml⁻¹ (R&D Systems), in a 37 °C and 5% CO₂ atmospheric environment. Npm1c/Flt3-ITD/Cas9 DM cells were passaged every 2 days and cultured for a short time (passages 3–5) to maintain the original leukemic properties.

Lara-Astiaso D., Goñi-Salaverri A., Mendieta-Esteban J., Narayan N., Del Valle C., Gross T., Giotopoulos G., Beinortas T., Navarro-Alonso M., Aguado-Alvaro L.P., Zazpe J., Marchese F., Torrea N., Calvo I.A., Lopez C.K., Alignani D., Lopez A., Saez B., Taylor-King J.P., Prosper F., Fortelny N, & Huntly B.J. (2023). In vivo screening characterizes chromatin factor functions during normal and malignant hematopoiesis. Nature Genetics, 55(9), 1542-1554.

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Generation of Bone Marrow-Derived Mast Cells from Knockout Mice

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WT (Cat# 000664, C57BL/6), Il6^−/− (Cat#002650, C57BL/6, N11) and Tnf^−/− (Cat#005540, B6/129S6, N1) mice were purchased from the Jackson Laboratories (Bar Harbor, ME). Congenic Tnf^−/− (C56BL/6, N10) mice were generated by back crossing to the C57BL/6 background. To prepare BMMCs, bone marrow cells from these mice were differentiated for 5 weeks in the presence of mouse IL3 (Cat#500-P53, PeproTech, Rocky Hill, NJ) and stem cell factor (Cat# AF-250–03, PeproTech) as described.^{16 (link),17 (link)} MC purity reached to >95%, which was confirmed with toluidine blue staining for the presence of metachromatic granules, and flow cytometric analysis for cell surface expression of CD117 (1:200, Cat#553356, BD Biosciences, San Jose, CA) and FcεR-1α (1:200, Cat#MCA6066F, Bio-Rad, Hercules, CA), but absence of Sca-1 (1:200, Cat#553108, BD Biosciences) expression.

He A., Fang W., Zhao K., Wang Y., Li J., Yang C., Benadjaoud F, & Shi G.P. (2019). Mast cell-deficiency protects mice from streptozotocin-induced diabetic cardiomyopathy. Translational research : the journal of laboratory and clinical medicine, 208, 1-14.

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Culturing B16F10, HEK-293T, and 32D Cells

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B16F10 cells, HEK-293T cells, and 32D clone 3 cells were purchased from the American Type Culture Collection (ATCC). B16F10 cells and HEK-293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, USA) supplemented with 10% heat-inactivated foetal bovine serum (Corning, USA). 32D clone 3 cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated foetal bovine serum and 10% mouse IL-3 (213-13; PeproTech, USA). B16F10 cells, HEK-293T cells, and 32D clone 3 cells were all cultured with 1% penicillin/streptomycin.

Han X., Luan T., Sun Y., Yan W., Wang D, & Zeng X. (2020). MicroRNA 449c Mediates the Generation of Monocytic Myeloid-Derived Suppressor Cells by Targeting STAT6. Molecules and Cells, 43(9), 793-803.

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In vitro Long-Term Hematopoietic Stem Cell Assay

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In vitro single LT-HSC culture assay was performed as described previously (Rathinam et al., 2011 (link)). In brief, we isolated LT-HSCs (Lin^–Sca-1⁺c-Kit⁺CD48^–CD150⁺) by flow cytometry and sorted single cells into 96-well plates. Single LT-HSCs were cultured in vitro in the presence of the following recombinant cytokines: mouse stem cell factor (50 ng/ml), mouse thrombopoietin (10 ng/ml), mouse IL-3 (10 ng/ml), mouse IL-6 (10 ng/ml), and human Flt3L (50 ng/ml; all from Peprotech). Cells were cultured in IMDM supplemented with 10% (volume/volume) FCS, 2 mM L-glutamine, 1% (volume/volume) penicillin-streptomycin, and 1 mM nonessential amino acids. 3 d later, cells were transferred into 24-well plates with 1 ml fresh medium containing cytokines for further culture. After 7-d cultures, cells were counted with a blood counting chamber or analyzed by flow cytometry.

Xiong Z., Xia P., Zhu X., Geng J., Wang S., Ye B., Qin X., Qu Y., He L., Fan D., Du Y., Tian Y, & Fan Z. (2019). Glutamylation of deubiquitinase BAP1 controls self-renewal of hematopoietic stem cells and hematopoiesis. The Journal of Experimental Medicine, 217(2), e20190974.

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MC38 Cell Lysate Primes Murine DCs

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RBC lysed bone marrow obtained from the femurs and tibias of C57BL/6 or β₂ microglobulin deficient mice (Jackson Laboratories Cat. 2087) were cultured with mouse IL-3 (PeproTech Cat. 213–13) and mouse FLT3 ligand (PeproTech Cat. 250–31L) both at 10 ng/mL each in RPMI + 10% FCS for 7 days to generate bone marrow derived dendritic cells. Separately, 10⁷ MC38 treated with splicing inhibitors vs. DMSO or expressing chicken ovalbumin were harvested, washed and resuspended in sterile PBS, and subjected to five cycles of rapid freeze-thaw (alternating between 37°C and dry ice/acetone) to generate a cell lysate. After brief centrifugation at 100xg, the soluble fraction in PBS was added to bone marrow derived DCs and left to incubate overnight for antigen phagocytosis in the presence of LPS (ThermoFisher Cat. 00–4976-93). DCs were subsequently washed three times to remove cell-free lysates and LPS and incubated in a 1:1 ratio with CFSE-labeled B6 splenic T cells (10⁵ stimulators with 10⁵ responders) as described above. The MLR was analyzed at day 5 by flow cytometry.

Lu S.X., De Neef E., Thomas J.D., Sabio E., Rousseau B., Gigoux M., Knorr D.A., Greenbaum B., Elhanati Y., Hogg S.J., Chow A., Ghosh A., Xie A., Zamarin D., Cui D., Erickson C., Singer M., Cho H., Wang E., Lu B., Durham B.H., Shah H., Chowell D., Gabel A.M., Shen Y., Liu J., Jin J., Rhodes M.C., Taylor R.E., Molina H., Wolchok J.D., Merghoub T., Diaz LA J.r., Abdel-Wahab O, & Bradley R.K. (2021). Pharmacologic modulation of RNA splicing enhances anti-tumor immunity. Cell, 184(15), 4032-4047.e31.

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Cell Culture Conditions for EGFR Studies

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mAb-108 expressing hybridoma cells (ATCC, HB-9764) were grown in high glucose DMEM media, no phenol red (Gibco, 31053044) supplemented with 10% fetal bovine serum (FBS – Gibco, 10270-106) and 1 mM sodium pyruvate (Gibco, 11360070). Chinese Hamster Ovary (CHO) cells (gift from Prof. Peter Parker at The Francis Crick Institute, UK) were grown in DMEM/F12 with no phenol red (Gibco, 21041-025) + 10% (v/v) FBS + 1% penicillin/streptomycin (Gibco, 15140148) and when made to stably express EGFR or mutants the media was supplemented with 4 µg/mL puromycin (Gibco, A1113803) to maintain expression. CHO cells expressing ΔC-EGFR (gift from Prof. Linda Pike, Washington University School of Medicine, USA) were grown in phenol-red-free DMEM supplemented with 10% FBS, 2 mM glutamine (Gibco, 25030081), 1% penicillin/streptomycin, 100 µg/mL hygromycin (Gibco, 10687010) and 100 µg/mL geneticin (Gibco, 10131035). Ba/F3 cells (Creative Biogene, CSC-C2045) stably expressing EGFR or EGFR mutants were grown in RPMI1640, no phenol red (Gibco, 11835063), with 2 mM L-glutamine and without HEPES + 10% heat-inactivated FBS (Gibco, 10500064) + 10 ng/mL mouse IL3 (Peprotech, 213-13) + 1% penicillin/streptomycin + 2 µg/mL puromycin. All cell lines were verified negative for mycoplasma before use and tested routinely.

Iyer R.S., Needham S.R., Galdadas I., Davis B.M., Roberts S.K., Man R.C., Zanetti-Domingues L.C., Clarke D.T., Fruhwirth G.O., Parker P.J., Rolfe D.J., Gervasio F.L, & Martin-Fernandez M.L. (2024). Drug-resistant EGFR mutations promote lung cancer by stabilizing interfaces in ligand-free kinase-active EGFR oligomers. Nature Communications, 15, 2130.

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Expansion and Transduction of Lin- BM Cells

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Mouse Lin⁻ BM cells were isolated and kept at −80°C overnight. Cells were thawed the following day and seeded in a 24-well plate at 1 × 10⁵ cells/well in StemSpan medium supplemented with complete growth medium supplement (CGMS; 1% penicillin/streptomycin, 50 ng/mL mouse SCF, 100 ng/mL FLT-3L, 100 ng/mL IL-11, and 20 ng/mL mouse IL-3; all from PeproTech) and incubated for 2 days. Plates coated with Retronectin (Takara Bio) were used to preload viral constructs, and the first round of transduction was completed with prestimulated Lin⁻ cells. A new Retronectin-coated plate was used for preloading viral vectors on the following day (day 0), and a second round of transduction was conducted by transferring transduced cells to the new plate. After overnight incubation, cells were transferred to 12-well plates and cultured in Iscove's modified Dulbecco's medium (IMDM; Biochrom) supplemented with CGMS. After day 15, cells were seeded at 100 cells/well in 96-well plates and cultured for an additional 2 weeks. Trypan blue exclusion counting was performed on days 1, 4, 6, 8, 11, and 15.

Huang J., Khan A., Au B.C., Barber D.L., López-Vásquez L., Prokopishyn N.L., Boutin M., Rothe M., Rip J.W., Abaoui M., Nagree M.S., Dworski S., Schambach A., Keating A., West M.L., Klassen J., Turner P.V., Sirrs S., Rupar C.A., Auray-Blais C., Foley R, & Medin J.A. (2017). Lentivector Iterations and Pre-Clinical Scale-Up/Toxicity Testing: Targeting Mobilized CD34+ Cells for Correction of Fabry Disease. Molecular Therapy. Methods & Clinical Development, 5, 241-258.

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Culturing Mouse and Human Leukemia Cells

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Mouse leukemia cells (CALM/AF10 and MLL/AF9) were cultured in Iscove’s Modified Dulbecco’s Media (IMDM) (Life Technologies) supplemented with 20% Fetal Bovine Serum (FBS) (Omega Scientific), 1% penicillin streptomycin (Life Technologies), and mouse SCF (20 ng/ml), mouse IL-3 (10 ng/ml) and mouse IL-6 (10 ng/ml) (all from PeproTech). Human leukemia cell lines (MOLM-13, OCI-AML3, MV4-11, and THP-1) were cultured in Roswell Park Memorial Institute (RPMI) medium (Life Technologies) with 10% FBS and 1% penicillin streptomycin.

Yamauchi T., Masuda T., Canver M.C., Seiler M., Semba Y., Shboul M., Al-Raqad M., Maeda M., Schoonenberg V.A., Cole M.A., Macias-Trevino C., Ishikawa Y., Yao Q., Nakano M., Arai F., Orkin S.H., Reversade B., Buonamici S., Pinello L., Akashi K., Bauer D.E, & Maeda T. (2018). Genome-wide CRISPR-Cas9 screen identifies leukemia-specific dependence on a pre-mRNA metabolic pathway regulated by DCPS. Cancer cell, 33(3), 386-400.e5.

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