Hoxb8-FL cells were transduced with pMSCV-MLL-ENL-IRES-eGFP retroviral vector. ME-Transformed cell line was then generated by serial re-plating on methylcellulose5 (link). Briefly, GFP-positive cells were FACS sorted, washed twice and seeded on methylcellulose as for colony-forming assays (see below). Colonies were recovered, washed and re-plated every 7 days. At the third re-plating, colonies were recovered, washed and transferred to liquid culture containing RPMI 1640 medium (Sigma-R8758) supplemented with 10% FBS (HyCloneTM GE Healthcare), 1% penicillin/streptomycin (Sigma-P0781), 1% l -Glutamine (Sigma-G7513), 0.1% 2-Mercaptoethanol (50 mM stock) (Gibco®), 10 ng/ml of recombinant murine interleukin 3 (IL-3) (PeproTech), 10 ng/ml of recombinant murine interleukin 6 (IL-6) (PeproTech) and 50 ng/ml of recombinant murine stem cell factor (SCF) (PeproTech). After two passages, SCF was removed from the media and following two more passages, IL-6 was also removed. Finally ME-Transformed cells were cultured long term in the presence of IL-3 only.
Recombinant murine stem cell factor scf
Manufactured by Thermo Fisher Scientific
Sourced in United States
Recombinant murine stem cell factor (SCF) is a laboratory reagent used in cell culture research. SCF is a cytokine that plays a crucial role in the regulation of hematopoietic stem and progenitor cells. This recombinant protein is derived from the mouse gene sequence.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Papain solution, by Worthington (2 mentions)
Recombinant murine il 3, by Thermo Fisher Scientific (2 mentions)
Fcεri, by BioLegend (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Recombinant murine interleukin 3 il 3, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Rmil 5, by R&D Systems (1 mentions)
Flt3l, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Antibiotic and antimycotic, by Merck Group (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
8 protocols using recombinant murine stem cell factor scf
1
Generation of ME-Transformed Cell Line
2
Murine Cortical and Amygdalar Neuron Isolation
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Neurons were obtained from the frontal cortical and amygdala regions mice 2–3 days postnatal that were aseptically dissected and cultured from each respective genotype (WT, Nf1+/−, Nf1+/−/Pak1−/−, Pak1−/−). Following dissection of each respective brain region, neuronal cells were isolated by dissociation both enzymatically and mechanically (via trituration through a flame-polished Pasteur pipette) in a Papain solution (12 units/ml; Worthington) as described previously41 . For this experiment, the neuronal cultures were assigned to one of two experimental conditions: (i) at basal levels and (ii) following the application of recombinant murine stem cell factor (SCF; PreproTech) at 10 ng/ml. SCF was applied to the neuronal cultures for 2 min. The cells were then washed with ice-cold PBS and lysed in buffer, as described below.
3
Isolation and Differentiation of Mouse Mast Cells
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Mouse mast cells (BMMC) were prepared from bone marrow cells isolated from femora, as described by Jensen et al. (42 (link)) with slight modifications. Briefly, femora were flushed with Hanks’ balanced salt solution, and the cell suspension was forced through a 70-µm mesh. Collected cells were resuspended in complete mast cell medium containing RPMI1640, 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 ng/ml recombinant murine IL-3, and 10 ng/ml recombinant murine stem cell factor (SCF; both from Peprotech, Hamburg, Germany), and cultured at 37°C in 5% CO2. Cells were transferred periodically to fresh flasks to remove adherent cells, and maintained for 4–6 weeks to allow mast cell differentiation. At this time, more than 96% of cells expressed the mast cell markers c-Kit (CD117) and Fc-epsilon receptor I, as determined by flow cytometry analysis.
4
Murine Bone Marrow Mast Cell Differentiation
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Bone marrow cells were collected from femurs from 4-to–8-month-old mice and cultured in Dulbecco's Modified Eagle Medium with glucose and L-glutamine, supplemented with 10% fetal bovine serum, penicillin/streptomycin, and sodium pyruvate (all from Gibco™, ThermoFisher Scientific, Waltham, MA, USA) plus 25 ng/mL recombinant murine stem cell factor (SCF) (all cytokines and growth factors were from Peprotech, Rocky Hill, NJ, USA) and 30 ng/mL interleukin (IL)-3. Bone marrow MCs were differentiated into either a MMCs with additional IL-9 at 5 ng/mL and transforming growth factor (TGF)-β at 1 ng/mL or a CTMC with IL-4 at 1 ng/mL. MCs were cultured in 75-cm2 tissue culture flasks, incubated at 37℃ in a humidified incubator under 5% (v/v) CO2 for a minimum of 4 weeks and up to 8 weeks before they were used for functional assays. Twice a week, the medium was changed by transferring the cell suspension to a 50-mL conical polypropylene centrifuge tube, and centrifuging for 10 minutes at 200×g, at room temperature. The culture flasks were changed every time the medium was changed. The maturity and purity of the cells were examined by flow cytometric analysis for the expression of c-Kit (eBioscience, San Diego, CA, USA) and FcεRI (Biolegend, San Diego, CA, USA).
5
ex vivo Culture and DQ-OVA Uptake in BmEOS
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The ex vivo culture of BmEOS was adapted from the previously published protocols with minor modifications (21 (link), 22 (link)). Briefly, bone marrow (BM) was flushed out from the femur and tibiofibula of C57bl/6J mice. After erythrocyte lysis, BM cells were seeded at 1×10^6/ml in IMDM media supplemented with 20% fetal bovine serum (Gibco), 1% penicillin/streptomycin, 2 mM L-glutamine (Gibco), 1 mM sodium pyruvate (Gibco), and 50 mM 2-mercaptoethanol (Sigma-Aldrich) in the presence of 100 ng/mL FLT-3L and 100 ng/mL recombinant murine stem cell factor (SCF, Peprotech), cultured from Day 0 to Day 4. On Day 4 and 8, the media containing SCF and FLT3-L was replaced with media containing 10 ng/mL recombinant mouse interleukin-5 (rmIL-5, R&D Systems) only. Every other day, from this point forward, media was replaced with fresh media containing rmIL-5. After Day 10, cells were seeded and used for subsequent experiments.
BmEOS were seeded in 48 wells plates and stimulated for 2 days with or without HDM and IL-25 in complete medium. To assess DQ-OVA uptake by BmEOS, BmEOS were treated with DQ-OVA for 12 h before being collected, washed, and further analyzed using flow cytometry. Cells cultured in the absence of DQ-OVA were set as a negative control.
BmEOS were seeded in 48 wells plates and stimulated for 2 days with or without HDM and IL-25 in complete medium. To assess DQ-OVA uptake by BmEOS, BmEOS were treated with DQ-OVA for 12 h before being collected, washed, and further analyzed using flow cytometry. Cells cultured in the absence of DQ-OVA were set as a negative control.
6
Murine Cortical and Amygdalar Neuron Isolation
7
Murine Mast Cell Culture Protocol
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Unfractionated peritoneal cells or cells obtained by hemiskull scraping were centrifuged at 300 g for 5 min at 4°C. The pellet was re-suspended in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS, 1% antibiotics (penicillin/streptomycin), 2 mM L-glutamine, 50 μM B-mercaptoethanol, 10 ng/ml murine recombinant stem cell factor (SCF; PeproTech, NJ, USA), and 10 ng/ml murine recombinant interleukin (IL)-3 (PeproTech, NJ, USA). After 2–3 weeks of culture, more than 98% of cells were identified as mast cells by Toluidine Blue staining. Cells were kept in culture for up to 5 weeks.
8
Murine Bone Marrow-Derived Mast Cell Isolation
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Bone marrow cells were obtained from femurs and tibias of 6–8-week-old C57BL/6 female and male mice. Cells were maintained in RPMI-1640 supplemented with 10% FBS, 5 μM β-mercaptoethanol (Life Technologies, USA) and 2% antibiotic and antimycotic (Sigma, USA) (complete RPMI 1640 medium), plus 10 ng/mL of murine recombinant IL-3 (Peprotech, USA) and 10 ng/mL of murine recombinant stem cell factor (SCF) (Peprotech, USA). Non-adherent cells were transferred to fresh culture medium twice a week for 6 − 9 weeks. The purity of bone marrow-derived mast cells (BMMC) was ≥ 90% as assessed by flow cytometry after staining of CD117 (clone: 2B8, BioLegend, USA; 0.25 μg/mL) and FcεRI (clone: MAR R-1, BioLegend, USA; 0.16 μg/mL).
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