Flt3 ligand

Manufactured by Miltenyi Biotec

Sourced in Germany, United States

The FLT3-ligand is a lab equipment product manufactured by Miltenyi Biotec. It is a recombinant protein that serves as a ligand for the FLT3 (Fms-like tyrosine kinase 3) receptor, which is expressed on the surface of certain hematopoietic stem and progenitor cells. The core function of the FLT3-ligand is to activate the FLT3 receptor, which plays a role in the proliferation and differentiation of these cells.

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Flt3-ligand (Flt3L) (5 citations) Human FLT3 ligand (4 citations) H-FLT3-ligand (3 citations)

8 protocols using flt3 ligand

Feeder-free CD34+ HSPC Expansion

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Fresh human CD34⁺ hematopoietic stem/progenitor cells (HSPC) isolated from cord blood from mixed donors were purchased from All Cells (Emeryville, CA). The in vitro feeder-free HSPC culture was modified based on a previous study (35 (link)). Specifically, CD34⁺ cells (1×10⁴cells/well in 96-well tissue culture plates) were cultured in RPMI-1640 media (Life Technologies) supplemented with 5% human AB serum (serum from human blood type AB donors; Valley Biomedical), 100 U/ml of penicillin (Life Technologies), 100 μg/ml of streptomycin (Life Technologies), and 50 μM 2-mercaptoethanol with the addition of IL-6 (25 ng/ml; Sigma Aldrich), Flt3 ligand (25 ng/ml; Miltenyi Biotec), and stem cell factor (SCF; 25ng/ml; Miltenyi Biotec). On day 7, half of the media was replaced with fresh media containing IL7 (20 ng/ml; Miltenyi Biotec), Flt3 ligand (25 ng/ml) and SCF (25 ng/ml). After day 14, cytokine-free media was used to replace half of the media weekly.
In all cases, cells were treated with TCDD (0.01, 0.1, 1 and 10 nM) or vehicle (VH, 0.02% DMSO) only on day 0 prior to addition of cytokines. In studies using AHR antagonist CH223191, cells were treated with antagonist 30 min prior to TCDD treatment.

Li J., Bhattacharya S., Zhou J., Phadnis-Moghe A.S., Crawford R.B, & Kaminski N.E. (2017). Aryl hydrocarbon receptor activation suppresses EBF1 and PAX5 and impairs human B lymphopoiesis. Journal of immunology (Baltimore, Md. : 1950), 199(10), 3504-3515.

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Generating T Cell Precursors from Murine Bone Marrow

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OP9-DL1, a mouse BM stromal cell line of (C57BL/6 x C3H)F₂-op/op origin transduced to express the Notch 1 ligand DLL1, was obtained under an MTA from J.-C. Zúñiga-Pflücker (University of Toronto, Toronto, Canada). Cell culture medium consisted of αMEM (Invitrogen) supplemented with 20% heat-inactivated FBS, 100 U/ml penicillin (Invitrogen), and 100 µg/ml streptomycin (Invitrogen). T cell precursors were generated in vitro as described previously with modifications (Min et al., 2002 (link)). In brief, LSK cells were sorted as described above and added to tissue culture–treated polystyrene 24-well tissue culture plates that were seeded with 4,000 OP9-DL1 cells per cm² the day before. The tissue culture media was supplemented with 10 ng/ml IL-7 (Miltenyi Biotec) and 10 ng/ml FLT3-ligand (Miltenyi Biotec) and different concentrations of Degarelix or vehicle (mannitol). Cultures were passaged every 4 d.

Velardi E., Tsai J.J., Holland A.M., Wertheimer T., Yu V.W., Zakrzewski J.L., Tuckett A.Z., Singer N.V., West M.L., Smith O.M., Young L.F., Kreines F.M., Levy E.R., Boyd R.L., Scadden D.T., Dudakov J.A, & van den Brink M.R. (2014). Sex steroid blockade enhances thymopoiesis by modulating Notch signaling. The Journal of Experimental Medicine, 211(12), 2341-2349.

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Culturing LSK Cells with DLL4

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LSK cells were cultured in non–tissue culture-treated polystyrene 24-well plates that were precoated with 5 µg/ml CH-296 fibronectin (Retronectin; Takara Bio. Inc.) and different concentrations of murine DLL4 (R&D Systems). Cell culture medium consisted of αMEM (Invitrogen) supplemented with 20% heat-inactivated FBS, 100 U/ml penicillin (Invitrogen), and 100 µg/ml streptomycin (Invitrogen). The media was supplemented with 5 ng/ml IL-7 (Miltenyi Biotec), 100 ng/ml FLT3-ligand (Miltenyi Biotec), and 100 ng/ml stem cell factor (SCF; Miltenyi Biotec) and changed every 4 d.

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Human CD34+ HSPCs Expansion in Engineered Niches

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Human CB CD34⁺ HSPC were extracted and isolated as indicated in Online Supplemental Methods. 7 × 10⁴ CD34⁺ cells were resuspended in Serum Free Expansion Medium (SFEM, Stemcell Technologies), supplemented with the following cytokines: SCF (10 ng/mL; cat# 130-093-991), FLT3-ligand (10 ng/mL; cat# 130-093-854); TPO (10 ng/mL; cat# 130-094-011) (all from Miltenyi Biotec.), and seeded in mature engineered niches for 24 h at a superficial velocity of 3000 μL/min. After the cell seeding phase, the superficial velocity was reduced to 300 μL/min for perfusion of the 1-week coculture. Culture medium was changed twice a week, during which the retrieved medium was harvested and spun down (300 g, 5 min) to collect cells in suspension. The pelleted cells were resuspended in fresh medium and injected back in the bioreactor.

Born G., Nikolova M., Scherberich A., Treutlein B., García-García A, & Martin I. (2021). Engineering of fully humanized and vascularized 3D bone marrow niches sustaining undifferentiated human cord blood hematopoietic stem and progenitor cells. Journal of Tissue Engineering, 12, 20417314211044855.

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HSPCs Expansion with MSC-EVs

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2.4 × 10⁵ CD34⁺ HSPCs were incubated with young or aged MSC-EVs collected from 1.2 × 10⁵ cells or XE buffer as control in CellGenix SCGM (CellGenix, Germany) with 10% vesicle-depleted FBS supplemented with SCF, Flt3 ligand and IL-3 (10 ng/ml each, all from Miltenyi Biotec, Germany). The EV samples were pooled beforehand. For vesicle depletion, the FBS was centrifuged with an Amicon-Ultra 100 kDa filter (Millipore, Germany) for 55 min at 3.000xg. Each sample was run in duplicates.
After the incubation, the cells were washed and counted by Countess II Automated Cell Counter (Thermo Fisher Scientific, Germany). Subsequently, samples were pooled and further analyzed using flow cytometry, clonogenic assays and RT-PCR.

Fichtel P., von Bonin M., Kuhnert R., Möbus K., Bornhäuser M, & Wobus M. (2022). Mesenchymal Stromal Cell-Derived Extracellular Vesicles Modulate Hematopoietic Stem and Progenitor Cell Viability and the Expression of Cell Cycle Regulators in an Age-dependent Manner. Frontiers in Bioengineering and Biotechnology, 10, 892661.

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Isolation and Polarization of Murine Macrophages

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Bone marrow was isolated from femora and tibiae of nude mice (20–25 g, Charles River Laboratory) by flushing the bones with 1 mL of Iscove's Modified Dulbecco's Media (IMDM, Sigma) containing 60% Fetal Clone II (FCII, Thermo Scientific) and 1 µg/mL P/S (Sigma). The marrow was passed through a 70 µm strainer and MΦ (M0) were selected and cultured in IMDM enriched with 15% FCII, 1 µg/mL P/S, 10 ng/mL recombinant mouse MΦ colony-stimulating factor (M-CSF, Miltenyi Biotec) and 10 ng/mL recombinant mouse Fms-related tyrosine kinase three ligand (Flt3-Ligand, Miltenyi Biotec) at 37°C in a humid atmosphere. M1 MΦ were obtained by culturing cells in 1 g/L glucose DMEM (Sigma) supplied with 15% FCII, 1 µg/mL P/S, 2 mM Gln (Sigma), 100 ng/mL LPS (Sigma) and 10 U/mL recombinant mouse IFNγ (eBioscience). M2 MΦ were obtained by culturing cells with 1g/L glucose DMEM supplemented with 15% FCII, 1% P/S, 2mM Gln (Sigma) and 50 ng/mL recombinant mouse IL4 (Miltenyi Biotec).

Leblond M.M., Gérault A.N., Corroyer-Dulmont A., MacKenzie E.T., Petit E., Bernaudin M, & Valable S. (2015). Hypoxia induces macrophage polarization and re-education toward an M2 phenotype in U87 and U251 glioblastoma models. Oncoimmunology, 5(1), e1056442.

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Oxidative Stress Induction in CD34+ Cells

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CD34+ cells were seeded in 24-well plates at 5 × 10⁵ cells/mL in Iscove’s modified Dulbecco’s medium (IMDM) (EuroClone, Pero, Milan, Italy) containing 10% Human Serum (Sigma-Aldrich, St. Louis, MO, USA), SCF (50 ng/mL), FLT3-ligand (50 ng/mL), TPO (20 ng/mL), IL-6 (10 ng/mL) and IL-3 (10 ng/mL) (all from Miltenyi Biotec, Bergish Gladbach, Germany), as previously described [29 (link)]. To induce oxidative stress, CD34+ cells were treated with Melittin, the main constituent and principal toxin of bee venom with powerful oxidant activity [30 (link)]. Briefly, CD34+ cells were seeded at 1 × 10⁶ cells/mL in IMDM medium supplemented with 10% Human Serum and Melittin 5 µg/mL (Sigma-Aldrich, St. Louis, MO, USA) for 6 and 24 h at 37 °C in a humidified atmosphere with 5% CO₂.

Genovese E., Mirabile M., Rontauroli S., Sartini S., Fantini S., Tavernari L., Maccaferri M., Guglielmelli P., Bianchi E., Parenti S., Carretta C., Mallia S., Castellano S., Colasante C., Balliu M., Bartalucci N., Palmieri R., Ottone T., Mora B., Potenza L., Passamonti F., Voso M.T., Luppi M., Vannucchi A.M., Tagliafico E, & Manfredini R. (2022). The Response to Oxidative Damage Correlates with Driver Mutations and Clinical Outcome in Patients with Myelofibrosis. Antioxidants, 11(1), 113.

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OP9-DL1 Coculture for T Cell Differentiation

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OP9-DL1 cells (kindly provided by J.C Zúniga-Pflücker, Sunnybrook Research Institute, Toronto) [27] were maintained at 75% confluency in Dulbecco's modified Eagles' medium (DMEM) with GlutaMAX (Invitrogen), 10% FCS, 1% penicillin and streptomycin, 50 µM βmercaptoethanol (Invitrogen), and 1% non-essential amino acids (Invitrogen) at 37°C and 5% CO2. Foetal thymocytes from WT or p110d -/-E15 thymic lobes were isolated and seeded onto a semi-confluent monolayer of OP9-DL1 cells cultured in flat-bottom 96-well plates with 200 µl of complete DMEM medium supplemented with 5 ng/ml Flt3 ligand (Miltenyi Biotec) and 1 ng/ml IL-7 (Miltenyi Biotec). IL-7 and Flt3 ligand were replenished every 3 days. Cells were cultured for 8 days at 37°C and 5% CO2, followed by analysis by flow cytometry.

Sumaria N., Martin S, & Pennington D.J. (2021). Constrained TCRγδ-associated Syk activity engages PI3K to facilitate thymic development of IL-17A-secreting γδ T cells. Science signaling, 14(692).

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