293T cells were cultured in DMEM (high glucose) medium containing 10% FBS. K562 cells were cultured in IMDM medium plus 10% FBS. Enriched fresh hematopoietic stem and progenitor cells (HSPCs) were cultured in vitro in Stemspan (STEMCELL Technologies) supplemented with 10 μg/ml heparin (Sigma), 10 ng/ml mouse SCF, 20 ng/ml mouse TPO, 20 ng/ml human IGF-II, 10 ng/ml mouse FGF-1, and 100 ng/ml human Angptl3 25 . All recombinant proteins were purchased from PEPROTECH or PROSPEC companies. All cell culture products were purchased from Life Technologies unless otherwise mentioned. All small molecule inhibitors were purchased from Cayman Chemicals.
Human igf 2
Manufactured by Merck Group
Human IGF-II is a recombinant protein that corresponds to the insulin-like growth factor-II (IGF-II) molecule found in humans. IGF-II is a member of the insulin-like growth factor family and plays a role in cell growth and development.
Automatically generated - may contain errors
Lab products found in correlation
Cell culture products, by Thermo Fisher Scientific (2 mentions)
Heparin, by Merck Group (2 mentions)
Small molecule inhibitors, by Cayman Chemical (2 mentions)
Mouse fgf 1, by Merck Group (2 mentions)
Mouse tpo, by Merck Group (2 mentions)
Mouse scf, by Merck Group (2 mentions)
Stemspan, by STEMCELL (2 mentions)
A83 01, by STEMCELL (1 mentions)
Penicillin streptomycin solution, by STEMCELL (1 mentions)
Methylcellulose, by Merck Group (1 mentions)
3 protocols using human igf 2
1
Culture and Expansion of HSPCs
2
Culture and Expansion of HSPCs
3
2D and 3D Colony Formation Assays
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For the 2D colony formation assay, 500 cells were cultivated in 6-well plates for 7-14 days. Colonies were stained with crystal violet (0.5% dissolved in 25% methanol). The number of colonies was counted using Fiji (Fiji, RRID:SCR_002285) as described (59, 60) .
For the 3D colony formation assay, 500 cells were cultured in 2 mL CnT-Prime Airway Epithelial Proliferation Medium (Cat. #CnT-PR-A; CELLNTEC) supplemented with 0.5% methylcellulose (Cat. #M0262; Sigma-Aldrich); 10ng/mL Human IGF-II (Cat. #100-12; PeproTech); 10ng/mL Human Heregulinβ-1 (Cat. #AF-100-03; PeproTech); 1 µM DMH-1 (Cat. #73632; STEMCELL Technologies); 1 µM A-83-01 (Cat. #72022; STEMCELL Technologies); 1% penicillin/streptomycin solution (Cat. #P0781, Sigma-Aldrich). in Nunclon Sphera 6-Well Plate (Cat. #174932; Thermo Fisher Scienti c). After 2-3 weeks, spheres were counted under the microscope, and images were taken at 40X magni cation.
For the 3D colony formation assay, 500 cells were cultured in 2 mL CnT-Prime Airway Epithelial Proliferation Medium (Cat. #CnT-PR-A; CELLNTEC) supplemented with 0.5% methylcellulose (Cat. #M0262; Sigma-Aldrich); 10ng/mL Human IGF-II (Cat. #100-12; PeproTech); 10ng/mL Human Heregulinβ-1 (Cat. #AF-100-03; PeproTech); 1 µM DMH-1 (Cat. #73632; STEMCELL Technologies); 1 µM A-83-01 (Cat. #72022; STEMCELL Technologies); 1% penicillin/streptomycin solution (Cat. #P0781, Sigma-Aldrich). in Nunclon Sphera 6-Well Plate (Cat. #174932; Thermo Fisher Scienti c). After 2-3 weeks, spheres were counted under the microscope, and images were taken at 40X magni cation.
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