Generation of BMDC and BMMC
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For BMMC induction, 1×106 BM cells were cultured supplemented with 5 ng/ml recombinant murine SCF and IL-3 for more than three weeks (>98% expressed c-kit and FcεRIα).
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Mast cell culture from bone marrow
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Mouse bone marrow-derived cultured mast cells (BMCMC) were differentiated from femoral bone marrow by culture in medium supplemented with recombinant mouse IL-3 and stem cell factor(rmSCF, , ) as described −/− bone marrow showed similar granular morphology, levels of active tryptase, and expression of FcεRIα and CD117, indicating that they mature similarly when cultured in the presence of IL-3 and SCF.
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Effects of overexpression of Bmi1 on HSCs in vitro.
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Single CD34-LSK cells were sorted into 96-well microtiter plates containing the SF-O3 medium supplemented with 10% FBS and multiple cytokines (10 ng/ml SCF, 10 ng/ml TPO, 10 ng/ml IL-3, and 3 u/ml EPO) and allowed to form colonies.
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Effects of overexpression of Bmi1 on HSCs in vitro.
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Single CD34-LSK cells were sorted into 96-well microtiter plates containing the SF-O3 medium supplemented with 10% FBS and multiple cytokines (10 ng/ml SCF, 10 ng/ml TPO, 10 ng/ml IL-3, and 3 u/ml EPO) and allowed to form colonies.
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Generation and visualization of neutrophil extracellular traps (NETs) from myeloid cell lines and human primary neutrophils.
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EPRO cells were generated by differentiating the EML multipotent progenitor cell line with ATRA, GM-CSF and IL-3.
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Induction of 2nd iPSCs
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All isolated somatic cells were cultured in the presence of Dox (2 ug/ml) for induction of 2nd iPSCs.
- Fibroblasts and hematopoietic cells were cultured in ES/iPSC medium.
- FLCD45 were cultured in the presence of 10 ng/ml human TPO, 10 ng/ml mouse EPO, 10 ng/ml mouse IL-3, 10 ng/ml mouse IL-6, 10 ng/ml mouse Flt3 ligand, 10 ng/ml mouse GM-CSF, 10 ng/ml mouse VEGF and 50 ng/ml mouse SCF .
- HSCs, HPCs and MPs were cultured in the presence of 10 ng/ml human TPO, 10 ng/ml mouse IL-3, 10 ng/ml mouse IL-6 and 10 ng/ml mouse Flt3 ligand .
- Macrophages were cultured in the presence of 5 ng/ml M-CSF .
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Induction of 2nd iPSCs
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All isolated somatic cells were cultured in the presence of Dox (2 ug/ml) for induction of 2nd iPSCs.
- Fibroblasts and hematopoietic cells were cultured in ES/iPSC medium.
- FLCD45 were cultured in the presence of 10 ng/ml human TPO, 10 ng/ml mouse EPO, 10 ng/ml mouse IL-3, 10 ng/ml mouse IL-6, 10 ng/ml mouse Flt3 ligand, 10 ng/ml mouse GM-CSF, 10 ng/ml mouse VEGF and 50 ng/ml mouse SCF .
- HSCs, HPCs and MPs were cultured in the presence of 10 ng/ml human TPO, 10 ng/ml mouse IL-3, 10 ng/ml mouse IL-6 and 10 ng/ml mouse Flt3 ligand .
- Macrophages were cultured in the presence of 5 ng/ml M-CSF .
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Biological activities of HoxB4 in Ba/F3 cells.
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2×103 Ba/F3-HoxB4 cells were cultured with 5 ng/ml IL-3 (survival cytokine) in 96-well plates for 3 days and cell proliferation activity was assessed via MTT assay.
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Lentiviral transduction of bone marrow cells
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Freshly isolated bone marrow (BM) cells were plated in Iscove's Modified Dulbecco's Medium (IMDM) (31980, , ) containing 5% fetal bovine serum (FBS), 1% penicillin/streptomycin, 200 mM glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 50 μM 2-mercaptoethanol, stem cell factor (SCF, 10 ng/ml, , ), Flt3-L (10 ng/ml), IL-11 (10 ng/ml), thrombopoietin (TPO, 10 ng/ml), IL-6 (10 ng/ml), and IL-3 (10 ng/ml).
- Bone marrow cells were spin-infected with 8 μg/ml polyprene and lentivirus (MOI of 0.1–0.5), at 2,500 rpm for 90 min.
- BM cells were then incubated for 3 hours at 37°C, counted and injected into NOD.SCID mice.
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Generation and isolation of MLL-AF9 leukemia cells
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MLL-AF9 mouse leukemias were generated in Dr. David Scadden's laboratory as described below.
- Actin-DsRED mice (JAX) were backcrossed for 10 generations onto C57BL/6J mice (JAX).
- These mice were sacrificed four days after injection with 150 mg/kg 5FU.
- BM cells were isolated from femurs and tibias, and red blood cells were lysed using ACK lysing buffer .
- Cells were incubated in RPMI supplemented with 20% FBS, 1% Pen-Strep, 6 ng/ml of IL-3 , 10 ng/ml of TPO , 10 ng/ml of IL-6 , at 37°C 5% CO2 overnight.
- MLL-AF9 was introduced by spin-infection (1 000 g for 90 minutes) using a retroviral vector (MSCV-MLL-AF9-neo) 6 live cells per mouse were injected into lethally irradiated (9Gy) C57BL/6 recipient mice 12 hours after viral infection.
- The mice were sacrificed once they became moribund.
- BM cells were isolated as described above, and subjected to ACK lysis.
- 200 000 cells were injected into sublethally irradiated (4.5 Gy)…
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MLL-AF9 mouse leukemias were generated in Dr. David Scadden's laboratory as described below.
- Actin-DsRED mice (JAX) were backcrossed for 10 generations onto C57BL/6J mice (JAX).
- These mice were sacrificed four days after injection with 150 mg/kg 5FU.
- BM cells were isolated from femurs and tibias, and red blood cells were lysed using ACK lysing buffer .
- Cells were incubated in RPMI supplemented with 20% FBS, 1% Pen-Strep, 6 ng/ml of IL-3 , 10 ng/ml of TPO , 10 ng/ml of IL-6 , at 37°C 5% CO2 overnight.
- MLL-AF9 was introduced by spin-infection (1 000 g for 90 minutes) using a retroviral vector (MSCV-MLL-AF9-neo) 6 live cells per mouse were injected into lethally irradiated (9Gy) C57BL/6 recipient mice 12 hours after viral infection.
- The mice were sacrificed once they became moribund.
- BM cells were isolated as described above, and subjected to ACK lysis.
- 200 000 cells were injected into sublethally irradiated (4.5 Gy) C57BL/6 recipients.
- Once these mice were moribund, ACK-lysed live BM cells were isolated as described above and 1×106 cells/mouse were used for intramuscular injections.
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