Cell culture
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Peripheral blood mobilized, purified human CD34+ cells were obtained from the Hematopoietic Cell Processing Core at the Fred Hutchinson Cancer Center as previously described
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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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MiR-221 is upregulated upon mast cell activation and its expression levels can be altered using lentivirus-based systems.
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A) Differentiated BMMCs were either left resting or were stimulated with 1.5 µg/mL IgE anti-DNP, 0.2 µg/mL DNP-HSA and 20ng/mL SCF prior analysis of miR-221 expression by TaqMan qRT-PCR.
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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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Model for iPGC emergence from SSEA1/Oct4+ clusters in EBs.
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Using a differential colony forming assay in the presence FGF2, SCF, LIF and RA which promotes survival and proliferation of PGCs, we show that RA-iPGC potential is highest in the cKitbright fraction and is absent in the cKitdim subpopulation of SSEA1+ cells.
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Expansion of CD34+ cells in serum free medium
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Isolated CD34+ cells were seeded at a density of 5×104cells per 500 µl of medium'>Stempro medium , in 24-well tissue culture plates .
- Growth factors used were IL-6, SCF, TPO, and Flt-3-L at a final concentration of 25 ng/ml with (test cells) and without (control cells) the addition of either zVADfmk −100 nM or zLLYfmk −10 µM (MP, , ).
- After the 10th day of culture, the cells were collected from the suspension culture, and centrifuged (1000 rpm for 5 minutes).
- The cells were used for assessing the in vitro homing properties or were used for in vivo homing studies in NOD/SCID mice.
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In vitro culture of Eos and MDSCs
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For obtaining Eos, naïve bone marrow cells were cultured in 10% medium'>FBS/PMI medium supplemented with stem cell factor and FLT3-ligand for 4 days, and then with medium containing recombinant mouse IL-5 .
- On day 12, the cells were collected for May-Grünwald-Giemsa staining and flow cytometry analysis6 cells per ml in 10% FBS/RPMI medium, supplemented with 40 ng ml−1 GM-CSF.
- On day 4, the cells were collected for FACS analysis of CD11b+ CD11c− Ly-6C+ Ly-6Glow MDSCs
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ES cell lines and EB differentiation
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Two days prior the onset of differentiation, ES cells were transferred to Iscove's Modified Dulbecco's Medium (IMDM,) containing the above components.
- EBs in suspension culture were then generated as described
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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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