Cells were seeded in low-attachment 96-well plates (CORNING) in decreasing serial dilutions (100-1 cells/well), in 200 μl of serum-free MEM-F12 (Sigma-Aldrich AB) supplemented with 25 ng/ml epidermal growth factor, 25 ng/ml basic fibroblast growth factor, 1×B27 (Thermofisher Scientific). Six technical replicates for each cell plating density were created. On day 10, the number of wells containing spheres was recorded and analyzed using the online ELDA analysis program (http://bioinf.wehi.edu.au/software/elda ) [33 (link)].
Mem f12
Manufactured by Merck Group
Sourced in United States
MEM-F12 is a cell culture medium used for the in vitro cultivation of a variety of cell types. It is a combination of Minimum Essential Medium (MEM) and Ham's F-12 Nutrient Mixture, providing a balanced formulation of amino acids, vitamins, inorganic salts, and other components to support cell growth and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Low attachment 96 well plates, by Corning (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Methylcellulose, by Merck Group (1 mentions)
96 well round bottom ultra low attachment microplates, by Corning (1 mentions)
Ea hy926, by Merck Group (1 mentions)
α mem, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
3 protocols using mem f12
1
Sphere Formation Assay of Tumor Cells
2
Mammosphere Formation Assay for Cancer Cells
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Mammosphere cultures of MDA-MB-231 and ZR-75-1 cells were performed using the hanging drop method by seeding 10 000 cells per drop in complete growth medium with 20% methylcellulose for 48 h. Mammospheres were collected and resuspended in MEM-F12 (Sigma-Aldrich AB) supplemented with 25 ng/ml epidermal growth factor, 25 ng/ml basic fibroblast growth factor, 1×B27 (Thermofisher Scientific) and 2% methylcellulose (Sigma-Aldrich AB). One mammosphere was placed per well in 96-well round bottom ultra-low attachment microplates (CORNING, Wiesbaden, Germany) and cultured for 5 days. Phase-contrast pictures were acquired every 24 h for 5 days. The cross-section area of the mammospheres was calculated using the Image-J software (National Institutes of Health, Bethesda, MD, USA).
3
In Vitro Cytocompatibility Evaluation
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Commercial cell lines were obtained from American Type Culture Collection (ATCC, Manassas, USA) and cultivated following the manufacturer's instructions. Accordingly, fibroblasts (human, HGF, PCS-201-018), mature osteoblasts (human, U2OS, HTB-96), fetal pre-osteoblasts (human, hFOB, CRL-11372) and endothelial cells (human, ea.hy926, CRL-2922) were selected to test specimens' cytocompatibility in vitro. U2OS and ea.hy926 were grown in Dulbecco's Modified Eagle Medium (DMEM, Sigma Aldrich) 10% FBS (Gibco, Invitrogen, USA) and 1% antibiotics, HGF were cultivated in alpha-modified Eagle's minimum essential medium (α-MEM, Sigma Aldrich) 10% FBS, 1% antibiotics while for hFOB a 50:50 mix of MEM and Ham's F12 (MEM/F12, 1:1,Sigma Aldrich) 10% FBS (Invitrogen, USA), 1% antibiotics and 3 mg/ml neomycin (G418, Sigma) was used. All cells were grown until a 90% confluence and then collected by enzymatic digestion (trypsin/EDTA) prior to each assay. Moreover, in order to preserve the original primary phenotype, HGF and hFOB were applied until passage 10.
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