Fms like tyrosine kinase 3 ligand flt3l

Manufactured by Thermo Fisher Scientific

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FMS-like tyrosine kinase 3 ligand (FLT3L) is a protein that binds to the FLT3 receptor, which is a member of the receptor tyrosine kinase family. FLT3L plays a role in the development and function of certain types of blood cells, including hematopoietic stem cells and dendritic cells.

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FMS-like tyrosine kinase 3 ligand FMS-like tyrosine kinase 3 Flt3L Tyrosine

9 protocols using fms like tyrosine kinase 3 ligand flt3l

Culturing Leukemia and Primary CD34+ Cells

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Human leukemia cell lines MV4-11 and Kasumi-1 were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA). MV4-11 and Kasumi-1 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% and 20% fetal bovine serum, respectively. Cell density was kept between 1×10⁵and 1×10⁶ cells/mL. Cells were cultured at 37 ℃ in a humid incubator supplied with 5% CO₂.
Primary CD34⁺ cells were isolated from cord blood with donors’ consent using the commercial isolation kit (STEMCELL Technologies, EasySep^TM Human CD34 Positive Selection Kit II, Canada). CD34⁺ cells were cultured in StemSpan SFEM II (STEMCELL Technologies, Vancouver, BC, Canada) supplemented with FMS-like tyrosine kinase 3 ligand (FLT3L; 100 ng/mL), thrombopoietin (100 ng/mL), and stem cell factor (100 ng/mL; PeproTech, East Windsor, NJ, USA). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). Informed consent was taken from all the patients. The study was approved by institutional ethics board of Shanghai Tenth People’s Hospital of Tongji University (22KN250).

Cheng Q., Xia J., Wang K., Zhang Y., Chen Y., Zhong Q., Wang X, & Wu Q. (2022). CRISPR/Cas9 ribonucleoprotein (RNP) complex enables higher viability of transfected cells in genome editing of acute myeloid cells. Annals of Translational Medicine, 10(16), 862.

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Generation of Murine Bone Marrow-Derived Eosinophils

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Bone marrow from naïve mice were used to generate bone marrow-derived eosinophils as previously described (10 (link)). Therefore, isolated bone marrow was counted using the CASY^® TT- cell counter system and cells were seeded in in Advanced RPMI medium with 20% FBS, 1% penicillin/streptomycin, 0.1% gentamycin, 2.5% HEPES and 1% Glutamax (Thermo Fisher Scientific GmbH, Germany). Cells were cultured with Stem cell factor (SCF) and FMS-like tyrosine kinase 3 ligand (FLT3L) (Peprotech, Rocky Hill, USA) for the first 4 days. Afterwards the growth factors were exchanged with IL-5 (Peprotech, Rocky Hill, USA). Half of the medium was exchanged every other day and on day 8, the cell culture flask was exchanged. After 12 days, cells were harvested and checked for the eosinophil purity using flow cytometry. A CytoFLEX Flow Cytometer (Beckman Coulter, Brea, USA) was used to analyze the purity of the eosinophils using anti-SiglecF-APC-Cy7 (BD Bioscience, San Jose, USA) antibodies.

Ehrens A., Rüdiger N., Heepmann L., Linnemann L., Hartmann W., Hübner M.P, & Breloer M. (2021). Eosinophils and Neutrophils Eliminate Migrating Strongyloides ratti Larvae at the Site of Infection in the Context of Extracellular DNA Trap Formation. Frontiers in Immunology, 12, 715766.

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Murine Hematopoietic Stem Cell Expansion

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HSCs expansion medium consists of BIT9500 (Stem Cell Technologies) supplemented with 50 ng/mL recombinant mouse (rm) SCF, 20 ng/mL FMS-like tyrosine kinase 3 ligand (Flt3L) and 10 ng/mL thrombopoietin (Tpo) (all from PeproTech, Inc.). p18SMI compounds were added where indicated. Bone marrow cells were harvested from C57BL/6 mice and made into a single-cell suspension. After immunomagnetic c-Kit enrichment (cKit-conjugated microbeads [MiltenyiBiotec, Bergisch-Gladbach]), cells were washed and resuspended in HSCs expansion medium. After culture, total nucleated cell counts were obtained, and a fraction of mononuclear cells (MNCs) or whole bone marrow cells were stained for flow cytometry analysis. Frequency of each cell population was determined by independently analyzing more than 5 × 10⁵ cells per sample in triplicate.

Xie X.Q., Yang P., Zhang Y., Zhang P., Wang L., Ding Y., Yang M., Tong Q., Cheng H., Ji Q., McGuire T., Yuan W., Cheng T, & Gao Y. (2015). Discovery of novel INK4C small-molecule inhibitors to promote human and murine hematopoietic stem cell ex vivo expansion. Scientific Reports, 5, 18115.

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Hematopoietic Stem Cell-Derived Dendritic Cells

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To generate hematopoietic stem cell-DCs (Stem-DCs), lineage-negative cells were selected from BM-MNCs by magnetic beads (MACS lineage depletion kit, Miltenyi Biotech, Germany) as HSCs. HSCs were seeded at a concentration of 1 × 10⁶ cells per well in 24-well plates and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, granulocyte–macrophage colony-stimulating factor (GM-CSF, 100 ng/mL), stem cell factor (SCF, 50 ng/mL), and FMS-like tyrosine kinase 3 ligand (Flt3L, 50 ng/mL) (Peprotech, Rocky Hill, NJ, USA) for 7–10 days to promote their proliferation and differentiation into the monocyte lineage. Then, the cells were placed in culture media containing GM-CSF (1000 U/mL) and interleukin (IL)-4 (1000 U/mL) (Peprotech, USA) for 4–5 days to induce their differentiation into DCs.

Lee S.W., Lee H., Lee K.W., Kim M.J., Kang S.W., Lee Y.J., Kim H, & Kim Y.M. (2023). CD8α+ dendritic cells potentiate antitumor and immune activities against murine ovarian cancers. Scientific Reports, 13, 98.

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Culturing K562 Cells and Primary AML Blasts

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K562 cells were purchased from ATCC (CCL-243 American Type Culture Collection [ATCC]). Cells were cultured using Iscove’s modified Dulbecco’s medium (IMDM; Thermo Scientific) supplemented with 1% penicillin/streptomycin (Life Technologies) and 10% fetal calf serum (FCS) (Biochrom).
Primary AML blasts were cultured as previously described.¹⁴ (link) In brief, blasts were thawed in IMDM +20% FCS + 100 μg/mL DNase I at 37°C and maintained in IMDM containing 15% BIT 9500 serum substitute (Stemcell Technologies), 100 ng/mL stem cell factor (Preprotech), 50 ng/mL FMS-like tyrosine kinase 3 ligand (FLT3L; Preprotech), 20 ng/mL interleukin (IL)-3 (Preprotech), 20 ng/mL granulocyte-colony stimulation factor (Preprotech), 50 ng/mL thrombopoietin (Preprotech), 10 μg/mL low-density lipoprotein (LDL) (Stem Cell Technologies), 0.1 mM β-mercaptoethanol (Thermo Scientific), 1% penicillin/streptomycin (Life Technologies), 0.5% ciprofloxacin, 500 nM stemregenin 1 (Focus Bioscience), 1 μM UM729 (kindly provided by Caroline Pabst), and 10 μL/mL Glutamax (Life Technologies) at 37°C, 5% CO₂. The study was reviewed and approved by the ethics committee of the physician’s chamber of Westfalen-Lippe and the medical faculty of the University of Muenster (2007-524-f-S and 2007-390-f-S) before the study began.

Brabetz O., Alla V., Angenendt L., Schliemann C., Berdel W.E., Arteaga M.F, & Mikesch J.H. (2017). RNA-Guided CRISPR-Cas9 System-Mediated Engineering of Acute Myeloid Leukemia Mutations. Molecular Therapy. Nucleic Acids, 6, 243-248.

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Murine Hematopoietic Stem Cell Isolation

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Fasudil was purchased from LC Laboratories (Woburn, MA). Busulfan, Cremaphor were purchased from Sigma-Aldrich (St. Louis, MO). Flourochrome-labeled monoclonal antibodies to CD45.1 and CD45.2 were purchased from BD Bioscience (San Jose, CA). The murine recombinant cytokines stem cell factor (SCF), thromobopoieitn (TPO) and FMS-like tyrosine kinase 3 ligand (FLT3L) were obtained from PeproTech (Rocky Hill, NJ). X-VIVO medium was from Lonza (Basel, Swiss). Antibodies to myosin light chain 2 (MLC) were purchased from Cell Signaling Technology (Danvers, MA).

William B.M., An W., Feng D., Nadeau S., Mohapatra B.C., Storck M.A., Band V, & Band H. (2015). Fasudil, a clinically-safe ROCK Inhibitor, Decreases Disease Burden in a Cbl/Cbl-b Deficiency-Driven Murine Model of Myeloproliferative Disorders. Hematology (Amsterdam, Netherlands), 21(4), 218-224.

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Generation of Mouse Bone Marrow-Derived Dendritic Cells

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Mouse bone marrow-derived dendritic cells (BMDCs) were prepared from C57BL/6J mice. Bone marrow cells were flushed from the femur and tibia with PBS and resuspended in RPMI-1640 complete medium containing 10% FBS, 2 mM L-glutamine, 1% antibiotic-antimycotic, 1% MEM nonessential amino acid solution, 1 mM sodium pyruvate, 10 mM HEPES supplemented with 100 ng/mL FMS-like tyrosine kinase 3 ligand (Flt3-L) (PeproTech) and 5 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) (PeproTech). On days three and six, a complete medium supplemented with 100 ng/mL Flt3-L and 5 ng/mL GM-CSF was added for further culture. On day nine, immature DCs were seeded at 2 × 10⁶ cells/well of a noncoated six-well plate and stimulated with CpG-2722 at 5 μg/ml for 24 h.

Tseng J.C., Yang J.X., Lee C.Y., Lo C.F., Liu Y.L., Zhang M.M., Huang L.R., Liu K.J., Wang C.C., Huang C.Y., Hong Y.R., Tsou L.K, & Chuang T.H. (2023). Induction of Immune Responses and Phosphatidylserine Exposure by TLR9 Activation Results in a Cooperative Antitumor Effect with a Phosphatidylserine-targeting Prodrug. International Journal of Biological Sciences, 19(9), 2648-2662.

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Generation of Lymphoid Cells from Sorted Cells

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For the generation of lymphoid cells, sorted cells were cultured in a T25 culture flask for 11–13 d on an OP9 or OP9-DL1 stromal cell layer in αMEM medium (Gibco) containing 10% FBS and 5 × 10⁻⁵ M 2-mercaptoethanol, supplemented with 50 ng/ml of stem cell factor (R&D Systems), 50 ng/ml of FMS-like tyrosine kinase 3 ligand (Flt3L; PeproTech), and 20 ng/ml of IL-7 (R&D Systems). Hematopoietic cells were harvested and analyzed for the expression of surface markers (B cells; B220⁺CD19⁺ cells, T cells; and CD4⁺CD8a⁺ cells) using flow cytometry.

Yokomizo T., Watanabe N., Umemoto T., Matsuo J., Harai R., Kihara Y., Nakamura E., Tada N., Sato T., Takaku T., Shimono A., Takizawa H., Nakagata N., Mori S., Kurokawa M., Tenen D.G., Osato M., Suda T, & Komatsu N. (2019). Hlf marks the developmental pathway for hematopoietic stem cells but not for erythro-myeloid progenitors. The Journal of Experimental Medicine, 216(7), 1599-1614.

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Bone Marrow-Derived Dendritic Cell Generation

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Bone marrow-derived dendritic cells (BMDCs) were generated from bone marrow of femurs and tibias of C57BL/6 mice. First, bone marrows were collected purging bones with PBS supplemented with Pen/Strep and 2% FBS. Then, red blood cells were lysed with 5 mL of lysis buffer (BioLegend). After filtration, through a 70 μm cell strainer, and washing, cells were resuspended in RPMI 1640 medium supplemented with Pen/Strep, 10 mM HEPES, 1% Non-Essential Amino Acids Solutions, 10% FBS, 50 nM β-mercaptoethanol, 20 ng/ml of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and 20 ng/ml of FMS-like tyrosine kinase 3 ligand (FLT3L) (Peprotech, Rocky Hill, New Jersey, USA) (complete medium) and seeded into 6-well plates at 7.5 × 10⁵/mL. After three days of culture, 1 mL of complete medium was added to each well and after three additional days, half of the culture medium was replaced. On day 7 BMDCs were harvested and resuspended at 2 × 10⁵/mL for co-culture.

Petrazzuolo A., Perez-Lanzon M., Martins I., Liu P., Kepp O., Minard-Colin V., Maiuri M.C, & Kroemer G. (2021). Pharmacological inhibitors of anaplastic lymphoma kinase (ALK) induce immunogenic cell death through on-target effects. Cell Death & Disease, 12(8), 713.

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