Flt3l

Manufactured by STEMCELL

Sourced in Canada

Flt3L is a recombinant human protein that supports the proliferation and differentiation of hematopoietic progenitor cells. It is a key growth factor for the expansion of dendritic cells and natural killer cells.

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Lab products found in correlation

Interleukin 3 (il 3), by STEMCELL (13 mentions) Stem cell factor (scf), by STEMCELL (9 mentions) Stemspan, by STEMCELL (8 mentions) Interleukin 6 (il 6), by STEMCELL (8 mentions) Stem cell factor (scf), by Thermo Fisher Scientific (5 mentions) Stem cell factor (scf), by R&D Systems (4 mentions) Lymphoprep, by STEMCELL (3 mentions) Interleukin 7 (il 7), by STEMCELL (3 mentions) Flt3l, by Thermo Fisher Scientific (3 mentions) Interleukin 3 (il 3), by Thermo Fisher Scientific (3 mentions) Il 23, by R&D Systems (2 mentions) Interleukin 7 (il 7), by R&D Systems (2 mentions) Rpmi 1640, by Thermo Fisher Scientific (2 mentions) L glutamine, by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Methocult 3234, by STEMCELL (2 mentions) Ack lysis buffer, by Lonza (2 mentions) P3 primary cell 4d nucleofector x kit, by Lonza (2 mentions) Dmpge2, by Cayman Chemical (2 mentions) Methocult h4434, by STEMCELL (2 mentions)

27 protocols using flt3l

Differentiation of CD34+ HSCs

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After 5 days of culture, the expanded cells were considered for further differentiation experiments. Where specified, expanded CD34⁺ HSCs were FACS sorted into different subsets including CD34⁺α4β7⁺, CD34⁺α4β7⁻, CD34⁺α4β7⁺CD48^+/− and CD34⁺α4β7⁺CD48⁺CD52^+/−. For up to 28 days of differentiation, cells were cultured in B0 differentiation media (as previously described (25 (link))), supplemented with SCF (20 ng/ml, R&D Systems), IL-3 (5 ng/ml, Stemcell), IL-7 (20 ng/ml, R&D), IL-15 (10 ng/ml, NIH), IL-23 (10 ng/ml, R&D) and Flt3L (10 ng/ml, Stemcell). In some experiments (specified in figure legends) cells were also cultured in the presence or absence of pre-plated and irradiated EL08.1D2 stromal cells. After a week of culture, IL-3 was excluded from the B0 differentiation media supplements. For plating progenitors on the stromal cells, 100 progenitor cells were plated per well of 96-well plates on the irradiated EL08.1D2 cells in 150 μl B0 differentiation media. Alternatively, cells were also plated without stroma using 96-well U-bottom plate and 1×10³ cells were cultured per well. Culturing, maintenance and preparation of irradiated stromal layer of EL08.1D2 cells on 96-well plate culture was as described earlier (55 (link)).

Tufa D.M., Yingst A.M., Trahan G.D., Shank T., Jones D., Shim S., Lake J., Winkler K., Cobb L., Woods R., Jones K, & Verneris M.R. (2020). Human Innate Lymphoid Cell Precursors Express CD48 that Modulates ILC Differentiation through 2B4 Signaling. Science immunology, 5(53), eaay4218.

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Culturing Human and Murine Hematopoietic Cells

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Human leukemia cell lines U937 (male, CALM10-AFF10) and MOLM13 (male, MLL-AF9, FLT3-ITD) were gifts from Dr. Ali Shilatifard at Northwestern University. Jurkat (male) cells were a gift from Dr. Panos Ntziachristos at Northwestern University. These cell lines were authenticated by short tandem repeat testing (IDEXX) prior to use, and cultured in RPMI-1640 (Gibco) supplemented with 10% FBS (Gibco), Penicillin/Streptomycin (Gibco), and LGlutamine (Gibco). CD34⁺ cells were obtained from Fred Hutchinson Cancer Center and cultured in StemSpan (StemCell Technologies) supplemented with 50 ng/ml SCF (StemCell), 10 ng/mL each of IL3, IL6, and Flt3L (StemCell Technologies). Primary murine hematopoietic stem and progenitor cells (HSPCs) were selected from flushed bone marrow of the hindlimbs using a CD117⁺ selection kit according to manufacturer’s protocol (StemCell Technologies). All primary HSPCs were maintained in StemSpan (StemCell Technologies) supplemented with 50 ng/mL recombinant murine SCF, 10 ng/mL recombinant murine IL3, 10 ng/mL recombinant murine IL6, and 1:200 human LDL. MLL-AF9 cells were maintained in RPMI-1640 (Gibco) supplemented with 10% FBS, L-glutamine, 100 ng/mL recombinant murine SCF, 50 ng/mL recombinant murine IL6, and 20 ng/mL recombinant murine IL3. All cells were cultured in 6-well flat-bottom plates at 37C with 5% CO₂.

Volk A., Liang K., Suraneni P., Li X., Zhao J., Bulic M., Marshall S., Pulakanti K., Malinge S., Taub J., Ge Y., Rao S., Bartom E., Shilatifard A, & Crispino J.D. (2018). A CHAF1B-dependent molecular switch in hematopoiesis and leukemia pathogenesis. Cancer cell, 34(5), 707-723.e7.

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Inducing Myeloid and Erythroid Differentiation

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Human CD34+CD38+ cells were maintained in SFEM (Stem Cell Technologies) enriched with CC100 cocktail of cytokines (SCF, 100 ng/ml; FLT3L, 100 ng/ml; IL-3, 20 ng/ml; IL-6, 20 ng/ml; Stem Cell Technologies) for 12 hr after thawing. Cells were then washed to eliminate the CC100 cytokines and were plated at a concentration of 10⁶ cells/ml in SFEM with the addition of either 100 ng/ml of G-CSF (Peprotech) or 10,000 U/ml of M-CSF (R&D Systems, USA) to induce myeloid differentiation, or 10 ng/ml of EPO (Peprotech) to induce erythroid differentiation. The cytokines were re-added at every medium-change.

Petruk S., Mariani S.A., De Dominici M., Porazzi P., Minieri V., Cai J., Iacovitti L., Flomenberg N., Calabretta B, & Mazo A. (2017). Structure of nascent chromatin is essential for hematopoietic lineage specification. Cell reports, 19(2), 295-306.

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Expansion and Differentiation of CD34+ HSCs

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After 4 days of expansion, the entire population was considered for further differentiation. In additional experiments, expanded CD34⁺ HSCs were FACS sorted based on PRLR expression into CD34⁺PRLR^- and CD34⁺PRLR⁺ populations. Expanded CD34⁺ HSCs, sorted CD34⁺PRLR⁻ or sorted CD34⁺PRLR⁺ cells were cultured in a previously described B0 differentiation media^{11 (link)} supplemented with SCF (20 ng/ml, R&D Systems), IL-3 (5 ng/ml, Stemcell) only for the first week, IL-7 (20 ng/ml, R&D Systems), IL-15 (10 ng/ml, NIH), IL-23 (10 ng/ml, R&D Systems) and FLT3L (10 ng/ml, Stemcell). PRL (1 ng/ml, Stemcell) was used to assess the effect of this hormone on CD56⁺ lineage differentiation, and SR-1 (1 µM, Cellagen Technologies) was used as a positive control to stimulate the generation of CD56⁺ lymphocyte development^{58 (link),59 (link)}. Granulocyte-monocyte colony-stimulating factor (GM-CSF, 1 ng/ml, Shenandoah Biotechnology) was used to differentiate granulocytes. Cells (1 × 10³ cells/per well) were cultured in a 96 well u-bottom plate. For the co-culture experiments, sorted CD34⁺PRLR⁻ and CD34⁺PRLR⁺ cells (in 2:1 ratio) were co-cultured in contact-dependent or with a trans-well system using flat-bottom 96 well plates. Cells were cultured for a total of up to 28 days of differentiation.

Tufa D.M., Shank T., Yingst A.M., Trahan G.D., Shim S., Lake J., Woods R., Jones K, & Verneris M.R. (2020). Prolactin Acts on Myeloid Progenitors to Modulate SMAD7 Expression and Enhance Hematopoietic Stem Cell Differentiation into the NK Cell Lineage. Scientific Reports, 10, 6335.

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Cytokine-Driven Expansion of CD34+ HSCs

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MSCs were seeded into 4 wells of a 6-well plate at an initial density of 1×10⁵ cells/well, and maintained in a humidified atmosphere of 5% CO₂ at 37℃ for 24 h. Umbilical cord blood-derived CD34⁺ cells (1×10⁵ cells/mL) were then plated onto the MSC layer in stem cell serum-free medium (Stem Cell Technologies, Vancouver, Canada); two wells were supplemented with 50 ng/mL of stem cell factor (SCF), Flt-3L (fms-like tyrosine kinase 3 Ligand), and thrombopoietin (TPO) (Stem Cell Technologies, Vancouver, Canada), and two wells were not. CD34⁺ HSCs (1×10⁵) were also cultured in two other wells in stem cell serum-free medium supplemented with 50 ng/mL of SCF, Flt-3L, and TPO under the same conditions. All cultures were performed in a humidified atmosphere of 5% CO₂ at 37℃ for 7 days. After 7 days of expansion, HSCs co-cultured with cytokines were separated into CD34⁺ and CD34⁻ fractions using a small MACs column (Miltenyi Biotec, Bergisch Gladbach, Germany).

Ahmadnejad M., Amirizadeh N., Mehrasa R., Karkhah A., Nikougoftar M, & Oodi A. (2017). Elevated expression of DNMT1 is associated with increased expansion and proliferation of hematopoietic stem cells co-cultured with human MSCs. Blood research, 52(1), 25-30.

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Expansion of UCB-Derived CD34+ HSCs

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Mononuclear cells were isolated by density gradient centrifugation using Lymphoprep (Stemcell). UCB-derived CD34⁺ HSCs were positively enriched using MACS CD34⁺ enrichment kit (Milteny). The cells (purity, >95%) were suspended (5×10⁴ cells/ml) in Stemspan II cell culture media (Stemcell) supplemented with 1% penicillin + streptomycin, stem cell factor (SCF, 100 ng/ml, R&D), FMS-like tyrosine kinase 3 (Flt3L, 100 ng/ml, Stemcell), thrombopoietin (TPO, 50 ng/ml, R&D) and low density lipoprotein (LDL, 10 μg/ml, Stemcell) and cultured in 24 well plates for 5 days of expansion. After 5 days of expansion the cells were expanded three-fold on average, while the proportion of CD34⁺ cells remained >95%.

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Hematopoietic Stem Cell Assay

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Bone marrow cells were aseptically isolated from 5-fluorouracil (5-FU; 150 mg/kg) treated C57BL/6 mice. After red blood cells were removed by using ACK lysis buffer (Lonza), 2 × 10⁴ nucleated cells were plated in triplicates into methylcellulose medium (MethoCult3234; Stem Cell Technologies) supplemented with 50 ng/mL FLT3L, 50 ng/mL SCF, 10 ng/mL IL3, 10 ng/mL IL6, and 10 ng/mL IL7 (Stem Cell Technologies). The colony numbers were counted every 7 days and re-plated for next round of serial replating.

Yoshida A., Choi J., Jin H.R., Li Y., Bajpai S., Qie S, & Diehl J.A. (2020). Fbxl8 suppresses lymphoma growth and hematopoietic transformation through degradation of cyclin D3. Oncogene, 40(2), 292-306.

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Assessing Total Hematopoietic Progenitor Cells

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For assessing the total hematopoietic progenitor cell activity, bone marrow was harvest from 5-fluorouracil treated C57BL/6 mice. After red blood cell lysis using ACK lysis buffer (Lonza), 2×10⁴ nucleated cells were plated in triplicates into methylcellulose medium (MethoCult 3234, Stem Cell Technologies) supplemented with 50 ng/ml Flt3L, 50 ng/ml SCF, 10 ng/ml IL-3, 10 ng/ml IL-6, and 10 ng/ml IL-7 (Stem cell Technologies). The colony number was counted 7 days after replating.

Li Y., Chitnis N., Nakagawa H., Kita Y., Natsugoe S., Yang Y., Li Z., Wasik M., Klein-Szanto A.J., Rustgi A.K, & Diehl J.A. (2015). PRMT5 is required for lymphomagenesis triggered by multiple oncogenic drivers. Cancer discovery, 5(3), 288-303.

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CD34+ Cell Expansion and Genetic Modification

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CD34+ cells were either freshly purified from human CB after obtaining informed consent and upon approval by the San Raffaele Hospital Bioethical Committee, or purchased frozen from Lonza. 10⁶ CD34+ cells/ml were stimulated in serum-free StemSpan medium (StemCell Technologies) supplemented with penicillin, streptomycin and human early-acting cytokines (for CB-derived cells: stem cell factor (SCF) 100 ng/ml, Flt3 ligand (Flt3-L) 100 ng/ml, thrombopoietin (TPO) 20 ng/ml, and interleukin 6 (IL-6) 20 ng/ml; for BM-derived cells: SCF 300ng/ml, Flt3-L 300 ng/ml, TPO 100 ng/ml, and IL-6 60 ng/ml; all purchased from Peprotech) for 24 or 48 hr and then infected with IDLVs at multiplicity of infection (MOI) 100-500. The following day the cells were electroporated with 175 μg/ml ZFNs encoding mRNAs (P3 Primary Cell 4D-Nucleofector X Kit, program EO-100; Lonza). For some experiments, the following drugs were supplemented to the culture media: 1 μM SR1 (kindly provided by T. Boitano and M. Cooke, GNF) added at every medium change, and 10 μM dmPGE2 (Cayman) added at the beginning of the culture, 1 hour before and just after electroporation. For CFC assays, 800 cells/plate were seeded one day after electroporation in methylcellulose-based medium (MethoCult H4434, StemCell Technologies). Two weeks after plating, colonies were counted and identified according to morphological criteria.

Genovese P., Schiroli G., Escobar G., Tomaso T.D., Firrito C., Calabria A., Moi D., Mazzieri R., Bonini C., Holmes M.C., Gregory P.D., van der Burg M., Gentner B., Montini E., Lombardo A, & Naldini L. (2014). Targeted Genome Editing in Human Repopulating Hematopoietic Stem Cells. Nature, 510(7504), 235-240.

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Notch Ligand-Driven Expansion of CD34+ Cells

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CB samples were processed with red blood cell lysis buffer and enriched for CD34⁺ cells using CliniMACS CD34 MicroBeads (Miltenyi Biotec, 130-017-501). CD34⁺ CB cells were then seeded onto plates coated with RetroNectin (5 μg/mL; Takara, T100A) plus Notch ligand Delta1 (2.5 μg/mL; ref. 39 (link)) overnight in SFEM II medium (StemCell Technologies, 09650FH) containing 50 ng/mL stem cell factor (SCF; StemCell Technologies, 78062), 50 ng/mL thrombopoietin (TPO; StemCell Technologies, 78210), and 50 ng/mL Fms-like tyrosine kinase 3 ligand (FLT3L; StemCell Technologies, 78009). Cells were transduced the following day with the C/G construct at an MOI of 200 or GFP control construct at MOI of 50. Transduced cells were grown on Notch ligand at 37°C in 5% CO₂ for 6 days and then sorted for GFP⁺ cells. Sorted GFP⁺ cells were either transplanted into NSG-SGM3 mice at 200,000 cells per mouse or placed in EC coculture or MC (see ref. 23 (link) and below) for long-term culture at 75,000 cells per well in a 6-well plate. In subsequent experiments using a CD34⁺ CB sample from another donor (CB 2, see Supplemental Figure 4), transduced cells were grown on Notch ligand for 2 days prior to placement in EC coculture or MC plating at 100,000 cells per well of a 12-well plate.

Le Q., Hadland B., Smith J.L., Leonti A., Huang B.J., Ries R., Hylkema T.A., Castro S., Tang T.T., McKay C.N., Perkins L., Pardo L., Sarthy J., Beckman A.K., Williams R., Idemmili R., Furlan S., Ishida T., Call L., Srivastava S., Loeb A.M., Milano F., Imren S., Morris S.M., Pakiam F., Olson J.M., Loken M.R., Brodersen L., Riddell S.R., Tarlock K., Bernstein I.D., Loeb K.R, & Meshinchi S. (2022). CBFA2T3-GLIS2 model of pediatric acute megakaryoblastic leukemia identifies FOLR1 as a CAR T cell target. The Journal of Clinical Investigation, 132(22), e157101.

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