Murine flt3l

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Murine Flt3L is a recombinant protein produced in E. coli. It is a growth factor that stimulates the proliferation and differentiation of hematopoietic progenitor cells.

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Flt3L (316 citations) Recombinant murine FLT3L (4 citations) Recombinant murine FLT-3 Ligand (FLT3L) (2 citations)

9 protocols using murine flt3l

Stat3 Activation in Dendritic Cells

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I3S, I3C, and DNFB were purchased from Sigma-Aldrich (St. Louis, MO, USA); murine Flt3L from eBioscience (San Diego, CA, USA). Antibodies used in the present study are: anti-signal transducer and activator of transcription 3 (STAT3) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-phospho-c-Src (Tyr416) from Cell Signaling Technology (Danvers, MA, USA); anti-phospho-STAT3 (Tyr705) from BD Biosciences (Franklin Lakes, NJ, USA); FITC anti-mouse CD11c, Alexa Fluor 647 anti-mouse B220, anti-ERK, and anti-phospho-ERK (Thr202/Tyr204) from Biolegend (San Diego, CA, USA). CpG oligodeoxyribonucleotides (CpG 2216: sequences are shown 5′-3′: small letters, phosphoorothioate linkage; capital letters, phosphodiester linkage 3′ of the base; bold, CpG-dinucleotides; ggGGGACGATCGTCgggggG) were manufactured by and purchased from Bioneer (Daejeon, Korea).

Hwang W.B., Kim D.J., Oh G.S, & Park J.H. (2018). Aryl Hydrocarbon Receptor Ligands Indoxyl 3-sulfate and Indole-3-carbinol Inhibit FMS-like Tyrosine Kinase 3 Ligand-induced Bone Marrow-derived plasmacytoid Dendritic Cell Differentiation. Immune Network, 18(5), e35.

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Dendritic Cell Derivation from Murine Bone Marrow

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Dendritic cells were derived from bone marrow cells with established protocols. Briefly, bone marrow was harvested from femurs and tibiae of BALB/c mice, treated with red-blood cells lysis buffer (BioLegend) and plated in R10 medium (RPMI-1640 supplemented with 10% heat-inactivated FBS, 1xAntiAnti, 12.5 mM HEPES, 4 mM Ultraglutamine) supplemented with either 20 ng/mL murine GM-CSF (BioLegend) or 200 ng/mL murine Flt3L (eBiosciences). Cells grown in GM-CSF were plated in 100 mm non-adherent petri dishes at a density of 2.5 × 10⁵ cells/mL in 10 mL, whereas cells grown in Flt3L in wells of 6-well-plates at a density of 1.5–2 × 10⁶ cells/mL. After 3 days, 5 mL R10 supplemented with 20 ng/mL murine GM-CSF were added to cultures grown in GM-CSF. Cells grown in GM-CSF were activated overnight at day 6, with 1 μg/mL LPS (Sigma), whereas cells grown in Flt3L were activated overnight at day 9, with 1 μg/mL poly I:C (GE Healthcare). Non-adherent cells were harvested, resuspended in R10 containing 10% DMSO and cryopreserved in liquid nitrogen.

Stringhini M., Spadafora I., Catalano M., Mock J., Probst P., Spörri R, & Neri D. (2021). Cancer therapy in mice using a pure population of CD8+ T cell specific to the AH1 tumor rejection antigen. Cancer Immunology, Immunotherapy, 70(11), 3183-3197.

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Establishing NOTCH1-induced T-ALL Model

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The NOTCH1-induced T-ALL model was established as previously decribed.^{17 (link)} Briefly, bone marrow cells were isolated from 8-week-old donor mice. Lineage negative (Lin^-) cells were enriched using a Lineage Cell Depletion Kit (Miltenyi Biotec), and pre-stimulated for 24 h in Dulbecco modified Eagle medium (Gibco) containing 20% fetal bovine serum (Gibco), 1% penicillin/streptomycin (Hyclone), 20 ng/mL murine FLT3-L, 20 ng/mL murine thrombopoietin and 100 ng/mL murine stem cell factor (PeproTech). Cells were then transduced with MigR1-ICN1 retroviruses and centrifuged in the presence of 6 Dg/mL polybrene. A second round of transduction was carried out on the next day. One million cells were then transplanted into sub-lethally (5.5 Gy) irradiated 8-week-old C57BL/6 female mice by tail vein injection.

Guo H., Xu J., Xing P., Li Q., Wang D., Tang C., Palhais B., Roels J., Liu J., Pan S., Huang J., Liu Z., Zhu P., Taghon T., Qing G., Van Vlierberghe P, & Liu H. (2023). RNA helicase DHX15 exemplifies a unique dependency in acute leukemia. Haematologica, 108(8), 2029-2043.

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Dendritic Cell Culture Protocols

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Bone marrow-derived DC were cultured as described previously (18 (link)). Briefly, femurs of Ex3^fl/fl, Ex3^DC−/−, or C57BL/6 mice were flushed with PBS and cultured for 9 days in media containing 10% fetal calf serum (Hyclone), 100 U/ml penicillin (Life Technologies), 0.1 mg/ml streptomycin (Life Technologies), 50 μM 2-mercaptoethanol (Sigma), and 20 ng/ml GM-CSF (Peprotech). Cells were harvested from the plates with gentle pipetting and cultured as indicated. Flt3L BMDC cultures were performed by flushing femurs with PBS, lysing red blood cells with ACK lysis buffer (Life Technologies), and plating cells in RPMI supplemented with 10% fetal calf serum, 25 mM HEPES (Life Technologies), 100 U/ml penicillin (Life Technologies), 0.1 mg/ml streptomycin (Life Technologies), and 100 ng/ml murine Flt3L (Peprotech). Cells were cultured for 9 days at 37°C. MutuDC1940 cells, kindly provided by Dr. Hans Acha-Orbea (University of Lausenne), were grown in a monolayer in media containing 8% fetal calf serum (Hyclone), 10 mM HEPES (Life Technologies), 50 μM 2-mercaptoethanol, 100 U/ml penicillin (Life Technologies), and 0.1 mg/ml streptomycin (Life Technologies). Cells were harvested by 10 min incubation with PBS and 5 mM EDTA.

Cohen S.B., Smith N.L., McDougal C., Pepper M., Shah S., Yap G.S., Acha-Orbea H., Jiang A., Clausen B.E., Rudd B.D, & Denkers E.Y. (2015). β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells. Journal of immunology (Baltimore, Md. : 1950), 194(1), 210-222.

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Murine Bone Marrow Isolation and Culture

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Briefly, bone marrow (BM) from murine tibias and femurs were passed through a 70 μm nylon mesh to remove debris. The isolated cells were treated with red blood lysis buffer. Total isolated bone marrow cells were then cultured in IMDM (Thermo Fisher; Grand Island NY) supplemented with 10% fetal bovine serum (FBS; Gibco), 2 mM L-glutamine (Gibco), 100 U/mL penicillin/streptomycin (Gibco) 200 ng/mL of murine Flt3-L (Peprotech) for 8–9 days in culture.

Elizondo D.M., Brandy N.Z., da Silva R.L., Haddock N.L., Kacsinta A.D., de Moura T.R, & Lipscomb M.W. (2019). Allograft Inflammatory Factor-1 Governs Hematopoietic Stem Cell Differentiation Into cDC1 and Monocyte-Derived Dendritic Cells Through IRF8 and RelB in vitro. Frontiers in Immunology, 10, 173.

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In Vitro B Cell Differentiation

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B cell populations were cocultured on MS5 stromal cells in a B cell medium composed of IMDM 10% FBS, 0.05 mM β-mercaptoethanol (Sigma-Aldrich), 2 mM L-glutamine (Invitrogen), penicillin (100 U/ml), and streptomycin (100 U/ml), and supplemented with 5 ng/ml of murine Il7, 10 ng/ml of murine Flt3L, and 10 ng/ml of murine stem cell factor (SCF) (Peprotech).

Fregona V., Bayet M., Bouttier M., Largeaud L., Hamelle C., Jamrog L.A., Prade N., Lagarde S., Hebrard S., Luquet I., Mansat-De Mas V., Nolla M., Pasquet M., Didier C., Khamlichi A.A., Broccardo C., Delabesse É., Mancini S.J, & Gerby B. (2023). Stem cell–like reprogramming is required for leukemia-initiating activity in B-ALL. The Journal of Experimental Medicine, 221(1), e20230279.

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Generation of Dendritic Cells from Mouse Bone Marrow

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pDCs were generated from BM of WT, pIII + IV^−/− and IDO^−/− mice as previously described [23] (link). Briefly, BM cells were recovered from tibia and femurs of mice and cultured, after red cell lysis, for 7 days in complete RPMI medium (10% heat-inactivated fetal bovine serum, 50 mM 2-βMercaptoethanol, 100 mM sodium Pyruvate and 100 μM of Penicillin/Streptomycin) supplemented with 100 ng/mL of murine Flt3L (PeproTech).
cDCs were generated as pDCs from BM of WT mice, by supplementing complete RPMI medium with 20 ng/ml granulocyte-macrophage colony-stimulating factor (PeproTech), and activated for 24 h with 5 ng/ml LPS (Enzo Life Sciences, Inc.).

Lippens C., Duraes F.V., Dubrot J., Brighouse D., Lacroix M., Irla M., Aubry-Lachainaye J.P., Reith W., Mandl J.N, & Hugues S. (2016). IDO-orchestrated crosstalk between pDCs and Tregs inhibits autoimmunity. Journal of Autoimmunity, 75, 39-49.

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Generation of cDCs and pDCs from Murine BM

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cDCs and pDCs were generated from BM of WT mice as previously described (18 (link)). Briefly, BM cells were recovered from tibia and femurs of mice and cultured, after red cell lysis, for 7–9 days in complete RPMI medium (10% heat-inactivated fetal bovine serum, 50 mm 2-βMercaptoethanol, 100 mm sodium Pyruvate and 100 μm of Penicillin/Streptomycin) supplemented with 100 ng/ml of murine Flt3L (PeproTech) for pDCs and with 20 ng/ml of GM-CSF for cDCs. For cDC culture, GM-SCF supplemented medium is added every 3 days and cells are split after 6 days of culture.

Lippens C., Garnier L., Guyonvarc'h P.M., Santiago-Raber M.L, & Hugues S. (2018). Extended Freeze-Dried BCG Instructed pDCs Induce Suppressive Tregs and Dampen EAE. Frontiers in Immunology, 9, 2777.

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Bone Marrow Cell Culture Protocol

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BM cultures were performed as described previously (Singh et al., 2013 (link)). In brief, 2 × 10⁵ cells of total nucleated BM cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) medium with 10% serum in presence of the indicated growth factors, 100 ng/ml murine SCF, 50 ng/ml murine IL6, 100 ng/ml murine TPO, and 100 ng/ml murine FLT3L (all cytokines form PeproTech, Rocky Hill, United States) for 10 days.

Liu Y., Chen Y., Deng X, & Zhou J. (2020). ATF3 Prevents Stress-Induced Hematopoietic Stem Cell Exhaustion. Frontiers in Cell and Developmental Biology, 8, 585771.

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