Flt3l

Manufactured by Amgen

Sourced in United States

Flt3L is a recombinant protein product produced by Amgen. It functions as a growth factor, stimulating the proliferation and differentiation of hematopoietic progenitor cells.

Automatically generated - may contain errors

Lab products found in correlation

Cpg b 1668, by Integrated DNA Technologies (2 mentions) Loxoribine, by InvivoGen (2 mentions) Stem cell factor scf, by Amgen (1 mentions) Facsdiva, by BD (1 mentions) Thrombopoietin, by Thermo Fisher Scientific (1 mentions) Cpg odn 1826, by InvivoGen (1 mentions) Aria 2, by BD (1 mentions) Automacs system, by Miltenyi Biotec (1 mentions) Foetal bovine serum (fbs), by Lonza (1 mentions) Collagenase d, by Roche (1 mentions)

5 protocols using flt3l

Expansion and Differentiation of Hematopoietic Stem Cells

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CB was obtained from the Royal North Shore Hospital and the Australian Cord Blood Bank. Ethical approval for the use of CB was obtained from the Human Research Ethics Committees of the relevant hospitals and the University of New South Wales (approval numbers: HREC 05188, NSCCH 0602-004M, SESIAHS 08/190). Bone marrow mononuclear cells were obtained from Lonza (Mt Waverly Australia). CB processing and isolation of CD34⁺ HSPC and GLYA⁺ cells are described in the Online Supplementary Methods. CD34⁺ HSPC were expanded for 1 week in Isocove modified Dulbecco media (Life Technologies, Carlsbad, CA, USA) with 20% fetal bovine serum (Trace Scientific, Melbourne, Australia), 100 ng/mL stem cell factor (SCF, Amgen, Thousand Oaks, CA, USA), 100 ng/mL thrombopoietin (Peprotech, Rocky Hill, NJ, USA), 100 ng/mL Flt-3 ligand (FLT-3L, Amgen) (STF), 50 mg/mL gentamycin and 200 mM glutamine. The cells were then cultured in cytokine combinations that force expansion and differentiation along specific lineages as described in our previous study (Online Supplementary Table S1).^{14 (link)} Differentiation was assessed by fluorescence activated cell sorting (FACS) analysis after staining cells with the conjugated antibodies detailed in Online Supplementary Table S2. Green fluorescent protein-positive (GFP⁺), GLYA⁺ and CD13⁺ subpopulations were purified by FACS using a FACS Diva (Becton Dickinson).

Richards L.A., Kumari A., Knezevic K., Thoms J.A., von Jonquieres G., Napier C.E., Ali Z., O’Brien R., Marks-Bluth J., Maritz M.F., Pickett H.A., Morris J., Pimanda J.E, & MacKenzie K.L. (2020). DKC1 is a transcriptional target of GATA1 and drives upregulation of telomerase activity in normal human erythroblasts. Haematologica, 105(6), 1517-1526.

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Dendritic Cell Differentiation from Bone Marrow

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Mice were sacrificed by cervical dislocation and bone marrow progenitors were washed out from bones (femurs and tibias). 2 × 10⁶ cells/mL were incubated in complete RPMI medium with 100 ng/mL Flt-3L (kindly provided by Amgen) in 6 wells-plates as described in de Brito et al.⁴⁷ (link) After 7 days, NP68 peptide (20 nM) with or without CpG ODN 1826 (2 μg/mL, InvivoGen) was added and cells were cultured overnight. The fraction of cDC (CD11c⁺, B220⁺) was measured by flow cytometry and was always >65%.

Wang S., Prieux M., de Bernard S., Dubois M., Laubreton D., Djebali S., Zala M., Arpin C., Genestier L., Leverrier Y., Gandrillon O., Crauste F., Jiang W, & Marvel J. (2024). Exogenous IL-2 delays memory precursors generation and is essential for enhancing memory cells effector functions. iScience, 27(4), 109411.

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Isolation and FACS purification of dendritic cells

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Bone marrow (BM) cells were isolated from femurs and tibias and a single cell suspension was prepared and cultured 7–8 days in the presence of 100ng/ml Flt3L (Amgen, Thousand Oaks, CA & Cell Dex Therapeutics, Needham, MA) as previously described (36 (link)). Spleens were incubated with 1mg/mL collagenase D for 20 min at 37°C and passed through a 100μm strainer to achieve a single cell suspension. Splenocytes were enriched with PanDC microbeads using an Automacs system (Miltenyi). PanDC⁺ fractions were stained with propidium iodide (PI) and FACS-purified using a BD ARIA II (BD) for pDCs (PI⁻CD11c^{intermediate/dim}CD11b⁻B220⁺PDCA⁺), and CD11b⁺ cDCs (PI⁻CD11c⁺B220⁻CD11b⁺) after B (CD19), T (Thy1.2) and NK (Nk1.1) cell exclusion. BM-pDCs and BM-cDCs were stained and sorted as PI⁻CD11c⁺CD11b⁻B220⁺PDCA⁺ and PI⁻CD11c⁺B220−CD11b⁺, respectively. Purity of the cells was > 92%. Cells were stimulated with CpG B 1668 (Integrated DNA Technologies, San Diego, CA) at 0.1μM (BM-derived DC) or 1μM (splenic DC), 10μM CpG A 2336 (Invivogen, San Diego, CA), and 100μM Loxoribine (Invivogen).

Macal M., Tam M.A., Hesser C., Di Domizio J., Leger P., Gilliet M, & Zuniga E.I. (2016). CD28 deficiency enhances type I interferon production by murine plasmacytoid dendritic cells. Journal of immunology (Baltimore, Md. : 1950), 196(4), 1900-1909.

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Isolation and Purification of Murine Immune Cells

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Mouse spleens were incubated with Collagenase D (1 mg ml⁻¹, Roche, Indianapolis, IN, USA) for 20 min at 37 °C, passed through a 100 μm strainer to obtain a single-cell suspension, and subsequently FACS purified using a BD ARIA (BD Biosciences) for B cells (CD19⁺), T cells (Thy1.2⁺), macrophages (Thy1.2⁻CD19⁻NK1.1⁻CD11c⁻CD11b⁺F4/80⁺), pDCs (Thy1.2⁻CD19⁻NK1.1⁻CD11c⁺CD11b⁻B220⁺PDCA⁺) and cDCs (Thy1.2⁻CD19⁻NK1.1⁻ and CD11c⁺B220⁻CD11b⁺ or CD11c⁺B220⁻CD8⁺).
BM cells were isolated from femurs and tibias and incubated in RPMI with 10% foetal bovine serum (Lonza, Walkersville, MD, USA) containing 100 ng ml⁻¹ of Flt3L (Amgen, Thousand Oaks, CA, USA) for 8 days. At day 8, BM-derived DC (BMDCs) were FACS purified for pDCs (CD11c^{intermediate/dim}CD11b⁻B220⁺PDCA⁺) and CD11b⁺ cDCs (CD11c⁺B220⁻CD11b⁺CD8⁻). Purity for all cell types was >95%.

Dallari S., Macal M., Loureiro M.E., Jo Y., Swanson L., Hesser C., Ghosh P, & Zuniga E.I. (2017). Src family kinases Fyn and Lyn are constitutively activated and mediate plasmacytoid dendritic cell responses. Nature Communications, 8, 14830.

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Differentiation of Murine Bone Marrow-Derived Dendritic Cells

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BM cells were isolated from femurs and tibias, and a single-cell suspension was prepared and cultured for 7 to 8 days in the presence of 100 ng/ml Flt3L (Amgen, Thousand Oaks, CA, and Cell Dex Therapeutics, Needham, MA), as previously described (73 (link)). The cells were stimulated with 0.1 μM CpG B 1668 (Integrated DNA Technologies, San Diego, CA), 100 ng/ml LPS, 25 μg/ml poly(I · C), and 100 μM loxoribine (Invivogen).

Harker J.A., Wong K.A., Dallari S., Bao P., Dolgoter A., Jo Y., Wehrens E.J., Macal M, & Zuniga E.I. (2018). Interleukin-27R Signaling Mediates Early Viral Containment and Impacts Innate and Adaptive Immunity after Chronic Lymphocytic Choriomeningitis Virus Infection. Journal of Virology, 92(12), e02196-17.

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