LAD-2 cells were cultured as described (Kirshenbaum et al., 2003 (link); Radinger et al., 2010 ) in StemPro-34 medium containing 13 ml of StemPro-34 Nutrient Supplement and l -glutamine (2 mM) and penicillin (100 U/ml)/streptomycin (100 μg/ml; all from GIBCO, Grand Island, NY) with 100 ng/ml recombinant human SCF added (Peprotech, Rocky Hill, NJ). Half of the medium supplemented with SCF was changed every 7 d. HLMCs were obtained by lung resection for bronchial carcinoma and purified using immunoaffinity magnetic selection using anti-CD117 (BD Biosciences, San Jose, CA) as described (Sanmugalingam et al., 2000 (link)) and were cultured as described (Cruse et al., 2008 (link)). All human subjects gave written informed consent, and the study was approved by the Leicestershire Research Ethics Committee, United Kingdom. Human peripheral blood–derived mast cells were cultured from CD34+ progenitors as described (Radinger et al., 2010 ). The donors provided an informed consent, and cells were obtained under a protocol (NCT00001756) approved by the National Institute of Allergy and Infectious Diseases, National Institutes of Health Internal Review Board.
Recombinant human scf
Manufactured by Thermo Fisher Scientific
Sourced in United States
Recombinant human SCF is a protein that functions as a growth factor. It is produced in a recombinant system and is suitable for use in cell culture and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant human il 3, by Thermo Fisher Scientific (4 mentions)
Stem pro 34 sfm media, by Thermo Fisher Scientific (3 mentions)
Hydrocortisone, by Merck Group (2 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Recombinant human il 6, by Thermo Fisher Scientific (2 mentions)
Recombinant human tpo, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Stempro 34 nutrient supplement, by Thermo Fisher Scientific (1 mentions)
Anti cd117, by BD (1 mentions)
Cd117, by Thermo Fisher Scientific (1 mentions)
Fc ri, by Thermo Fisher Scientific (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
West pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Amaxa transfection kit kit 5, by Lonza (1 mentions)
Dnp specific mouse ige spe 7, by Thermo Fisher Scientific (1 mentions)
Compound 48 80, by AnaSpec (1 mentions)
P nitrophenyl n acetyl β d glucosamine pnag, by Merck Group (1 mentions)
14 protocols using recombinant human scf
1
Culture and Purification of Mast Cells
2
Culture and Characterization of LAD2 Cells
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The LAD2 cell line (a gift from Arnold Kirshenbaum and Dean Metcalfe from the National Institutes of Health, NIAID) was cultured in StemPro-34 SFM media (Life Technologies, Burlington, ON, Canada) supplemented with 2mM L-glutamine, 100 U/ml penicillin, 50 mg/ml streptomycin, and 100 ng/ml recombinant human SCF (Peprotech, Rocky Hill, NJ, USA). Cells were maintained at 1×105 cells/ml at 37°C and 5% CO2 and periodically tested for the expression of CD117 (eBioscience, Invitrogen Carlsbad, CA, USA), and FcϵRI (eBioscience, Invitrogen Carlsbad, CA, USA) by flow cytometry using a CytoFlex flow cytometer (Beckman Coulter, Brea, CA, USA).
3
MRGPRX2-Mediated Mast Cell Activation
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All cell culture reagents and DNP-specific mouse IgE (SPE-7) were purchased from Invitrogen (Carlsbad, CA, USA). Recombinant murine interleukin-3 (IL-3), stem cell factor (SCF), and recombinant human SCF were purchased from Peprotech (Rocky Hill, NJ, USA). DNP-BSA and p-nitrophenyl-N-acetyl-β-D -glucosamine (PNAG) were from Sigma-Aldrich (St. Louis, MO, USA), Compound 48/80 was from AnaSpec (Fremont, CA, USA). Amaxa transfection kit (Kit V) was purchased from Lonza (Gaithersburg, MD, USA). PE anti-human MRGPRX2 antibody was purchased from Biolegend (San Diego, CA, USA). Polyclonal MRGPRX2 Ab was purchased from Novus Biologicals (Littleton, CO, USA). HRP-conjugated anti-rabbit IgG was from Cell Signaling Technologies (Danvers, MA, USA). West Pico Chemiluminescent Substrate was from Thermo Scientific (Rockford, IL, USA). DNeasy Blood and Tissue Kit was purchased from Qiagen (Germantown, MD, USA). QuikChange II Site-Directed Mutagenesis Kit was purchased from Agilent Genomics (Santa Clara, CA, USA). Plasmid encoding hemagglutinin (HA)-tagged human MRGPRX2 in pReceiver-MO6 vector was obtained from GeneCopoeia (Rockville, MD, USA). Antimicrobial peptidomimetics (compound 1, compound 2, compound 3, compound 4, and compound 5) were obtained from Fox Chase Chemical Diversity Center (Doylestown, PA, USA).
4
Culturing Human Mast Cell Lines
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HMC-1.1 and HMC-1.2 human mast cell lines were cultured in Iscove's Modified Dulbecco's Medium (Lonza, Mississauga, ON, Canada) containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were maintained at 1 × 105 cells/ml at 37°C and 5% CO2. The LAD2 cell line was cultured in StemPro-34 SFM media (Life Technologies, Burlington, ON, Canada) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 50 mg/ml streptomycin, and 100 ng/ml recombinant human SCF (Peprotech, Rocky Hill, NJ). Cells were maintained at 1 × 105 cells/ml at 37°C and 5% CO2 and periodically tested for expression of KIT and FcεRI by flow cytometry. The cells were used within 8 weeks of thawing from cryopreservation.
5
Culturing Primary Mouse Fibroblasts and Hematopoietic Cell Lines
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Primary mouse embryonic fibroblasts MEFs were generated and cultured according to standard protocols [45 (link)]. The Erythroid Myeloid Lymphoid (EML) cell line described previously [22 (link)] was maintained in Iscove's Modified Dulbecco's Medium (IMDM), 20% fetal bovine serum (FBS), Glutamax, penicillin-streptomycin supplemented with 100 ng/mL recombinant murine SCF (Peprotech). Human cell lines K562 [46 (link)] and M07e [27 (link)] were maintained in RPMI-1640 with glutamine and HEPES (Invitrogen), 10% FBS, penicillin-streptomycin. M07e were supplemented with 100 ng/mL recombinant human SCF and 30 ng/mL GM-CSF (Peprotech).
6
Hematopoietic Growth Factor Protocol
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Recombinant human SCF, IL-3 and EPO were from Peprotech (Hamburg, Germany). Insulin solution, holo-transferrin, heparin, hydrocortisone, bovine albumin, crystal violet, and DEAE-Dextran were from Sigma/Aldrich (Schnelldorf, Germany). MgCl2, CaCl2, NaHCO3, and formaldehyde were from Carl Roth (Karlsruhe, Germany).
7
Culturing LAD2 Human Mast Cells
8
Erythroid Differentiation of Transfected CD34+ HSPCs
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Transfected CD34+ HSPCs were differentiated into mature RBCs using a three-phase erythroid differentiation protocol, as previously described12 (link),65 (link). During the first phase (day 0 to day 6), cells were cultured in a basal erythroid medium supplemented with 100 ng/ml recombinant human SCF (PeproTech), 5 ng/ml recombinant human IL-3 (PeproTech), 3 IU/ml EPO Eprex (Janssen-Cilag) and 10−6 M hydrocortisone (Sigma). During the second phase (day 6 to day 9), cells were co-cultured with MS-5 stromal cells in the basal erythroid medium supplemented with 3 IU/ml EPO Eprex (Janssen-Cilag). During the third phase (day 9 to day 20), cells were co-cultured with stromal MS-5 cells in a basal erythroid medium without cytokines. Heat-inactivated human AB serum was added during the third phase of the differentiation (10%; day 13 to day 20). Erythroid differentiation was monitored by flow cytometry analysis of CD36, CD71, GYPA, BAND3, and α4-Integrin erythroid surface markers and of enucleated cells using the DRAQ5 double-stranded DNA dye. 7AAD was used to identify live cells. The gating strategy used to assess erythroid surface markers and enucleated cells is shown in Supplementary Fig. 19 .
9
High-Throughput Screening of MLL-AF9;FLT3-ITD Cells
10
Generation of Human Mast Cells from CD34+ Cells
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Two hundred milliliter whole blood was drawn from each participant by venipuncture. Human mast cells were generated as previously described 9 , 13 . In brief, peripheral blood mononuclear cells were purified by lymphoprep™ density gradient separation (Axis‐Shield PoC, Oslo, Norway) and CD34+ hematopoietic stem cells were isolated from peripheral blood mononuclear cells by magnetic‐activated cell sorting using CD34 MicroBead Kit (Miltenyi Biotec, GmbH, Bergisch Gladbach, Germany) according to the manufacturer's instruction. Enriched CD34+ cells were cultured in StemPro®‐34 medium (Invitrogen, Carlsbad, CA) supplied with 100 ng/ml recombinant human SCF (PeproTech, Rocky Hill, NJ), 100 ng/ml recombinant human IL‐6 (PeproTech), 100 U/ml penicillin (Sigma–Aldrich, St Louis, MO), 100 μg/ml streptomycin and 2 mM L‐glutamine (Sigma–Aldrich). Recombinant human IL‐3 (30 ng/ml) (PeproTech) was included in the medium for the first week only. After 7–8 weeks of culture, the PBdMC were used for experiments. The purity of cultured PBdMC was assessed by toluidine blue staining and flow cytometry analysis and revealed >90% metachromatic stained cells and CD117+FcϵRI+ cells.
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