To analyze the differentiation potential of MMPs, single MPP cells were sorted by MoFlo XDP (Beckman Coulter) and plated (one cell/well) in the MEM alpha (Life technology) medium containing 20% ES cell-grade FBS (Millipore), SCF (50 ng/ml: BioLegend), and Flt3 ligand (30 ng/ml: BioLegend) on the layer of OP9 cells in 96-well plates as previously described33 (link). The OP9 cell line was obtained from ATCC and confirmed to be free of mycoplasma by using the MycoAlert PLUS kit (Lonza). Two days after cell plating, IL-3 (10 ng/ml; BioLegend), IL-7 (10 ng/ml; BioLegend), and GM-CSF (10 ng/ml; R&D Systems) were added to each well to induce cell differentiation. “Plating efficiency” denotes cells successfully expanded in culture, determined by microscopic observation. Expanded cells were analyzed by flow cytometry to determine whether they developed into either granulocyte/macrophages (GM) (Gr-1+CD11b+B220−CD19− and Gr-1−CD11b+B220−CD19−) or B cells (Gr-1−CD11b−B220+CD19+).
Flt3 ligand
Manufactured by BioLegend
Sourced in United States, Canada
Flt3 ligand is a recombinant protein that functions as a growth factor. It binds to the Flt3 receptor on the surface of certain cell types, promoting their proliferation and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Stem cell factor (scf), by BioLegend (4 mentions)
Interleukin 7 (il 7), by BioLegend (2 mentions)
Mycoalert plus kit, by Lonza (2 mentions)
Es cell grade fbs, by Merck Group (2 mentions)
Interleukin 3 (il 3), by BioLegend (2 mentions)
Moflo xdp, by Beckman Coulter (2 mentions)
Alpha mem, by Thermo Fisher Scientific (2 mentions)
Gm csf, by R&D Systems (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Ab serum, by Valley Biomedical (1 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
X vivo 20, by Lonza (1 mentions)
N acetyl l cysteine, by Thermo Fisher Scientific (1 mentions)
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by BioLegend (1 mentions)
Il 15, by Thermo Fisher Scientific (1 mentions)
Cd11b, by BioLegend (1 mentions)
4 protocols using flt3 ligand
1
Differentiation potential of single MPP cells
2
Differentiation potential of single MPP cells
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To analyze the differentiation potential of MMPs, single MPP cells were sorted by MoFlo XDP (Beckman Coulter) and plated (one cell/well) in the MEM alpha (Life technology) medium containing 20% ES cell-grade FBS (Millipore), SCF (50 ng/ml: BioLegend), and Flt3 ligand (30 ng/ml: BioLegend) on the layer of OP9 cells in 96-well plates as previously described33 (link). The OP9 cell line was obtained from ATCC and confirmed to be free of mycoplasma by using the MycoAlert PLUS kit (Lonza). Two days after cell plating, IL-3 (10 ng/ml; BioLegend), IL-7 (10 ng/ml; BioLegend), and GM-CSF (10 ng/ml; R&D Systems) were added to each well to induce cell differentiation. “Plating efficiency” denotes cells successfully expanded in culture, determined by microscopic observation. Expanded cells were analyzed by flow cytometry to determine whether they developed into either granulocyte/macrophages (GM) (Gr-1+CD11b+B220−CD19− and Gr-1−CD11b+B220−CD19−) or B cells (Gr-1−CD11b−B220+CD19+).
3
Culturing 293T, T-cells, and CD34+ Cells
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293T cells were maintained in Dulbecco’s Modification of Eagle’s Medium supplemented with glutamax, non-essential amino acids, penicillin/streptomycin and 10% fetal bovine serum ThermoFisher (Waltham, MA, USA). Cells were maintained at 37 °C and 5% CO2.
T-cells were grown in X-VIVO-20 (Lonza, Allendale, NJ, USA) with 10% AB serum (Valley Biomedical, Winchester, VA, USA), 300 IU of IL-2 and 5 ng/mL each of IL-7 and IL-15 (PeproTech, Rocky Hill, NJ, USA), N-Acetyl-l -Cysteine, penicillin/streptomycin, and Gluta-MAX-I each from ThermoFisher (Waltham, MA, USA).
CD34+ hematopoietic stem cells were cultured in StemSpan SFMII media containing 1 µM SR1 each from STEMCELL Technologies, Cambridge, MA, USA and human cytokines Flt-3 ligand (100 ng/mL), SCF (100 ng/mL), TPO (100 ng/mL), IL-6 (100 ng/mL) all from Biolegend, San Diego, CA, USA.
T-cells were grown in X-VIVO-20 (Lonza, Allendale, NJ, USA) with 10% AB serum (Valley Biomedical, Winchester, VA, USA), 300 IU of IL-2 and 5 ng/mL each of IL-7 and IL-15 (PeproTech, Rocky Hill, NJ, USA), N-Acetyl-
CD34+ hematopoietic stem cells were cultured in StemSpan SFMII media containing 1 µM SR1 each from STEMCELL Technologies, Cambridge, MA, USA and human cytokines Flt-3 ligand (100 ng/mL), SCF (100 ng/mL), TPO (100 ng/mL), IL-6 (100 ng/mL) all from Biolegend, San Diego, CA, USA.
4
Isolation and Stimulation of Murine Lineage-Negative Cells
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Total bone marrow cells were isolated from the tibias and femurs of 6- to 10-week-old male C57B6/J and Il2rg-mutated mice. Then, the cells were treated with ammonium-chloride-potassium (ACK) lysing buffer to remove erythrocytes, followed by magnetic bead selection of Lin− fractions using antibodies against Gr-1, CD11b, B220 (CD45R), Ter119, and CD3 (BioLegend, San Diego, CA, USA). Isolated Lin− cells were stimulated for 24 h in Iscove's modified Dulbecco's medium (IMDM) medium supplemented with insulin–transferrin–selenium (ITS) supplement-A (STEMCELL Technologies, Vancouver, BC, Canada), 100 ng/mL murine thrombopoietin (BioLegend), 100 ng/mL stem cell factor (BioLegend), and 100 ng/mL Flt3-ligand (BioLegend) before electroporation.
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