Single cells were plated at 0.5 x 103 cells/well in ultra-low attachment plates (Corning) in a serum-free DMEM/F12 phenol-free medium (Corning) containing 1% methylcellulose (Sigma), 1% B27 supplement (Invitrogen), 1% penicillin-streptomycin, 10 μg/mL heparin (Sigma Aldrich #H3149), 20 ng/mL EGF (Sigma Aldrich #E9644) and 20 ng/mL basic fibroblast growth factor (bFGF; Thermo Fisher Scientific #PHG0024). To generate secondary spheroids after 7-10 days, primary spheres were collected and dissociated enzymatically in 0.25% trypsin-EDTA (Corning). Single cells were plated in the media described above supplemented with complete culture medium (1:1 ratio). Spheroids were allowed to grow for 7-10 days. Four representative images/well were taken and spheroids larger than 50 μm were scored by manual counting. To measure tumorsphere size, 30-40 tumorspheres/condition were traced in ImageJ.
Dmem f12 phenol free medium
Manufactured by Corning
DMEM/F12 phenol-free medium is a cell culture medium formulation used for the in vitro cultivation of a variety of cell types. It is a modification of the original DMEM/F12 medium, with the phenol red indicator dye removed. This phenol-free formulation is designed to provide a reliable, consistent, and defined growth environment for sensitive cell lines and applications where the presence of phenol red may interfere with experimental results or cell behavior.
Automatically generated - may contain errors
Lab products found in correlation
Ultra low attachment plate, by Corning (2 mentions)
Methylcellulose, by Merck Group (2 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Corning (1 mentions)
Basic fgf, by Thermo Fisher Scientific (1 mentions)
40 μm sieve, by BD (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Heparin, by Merck Group (1 mentions)
2 protocols using dmem f12 phenol free medium
1
Tumorsphere Assay for Stem-like Cell Enrichment
2
Tumorsphere Culture and Passaging
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A single-cell solution was obtained after enzymatic dissociation in 0.25% trypsin-EDTA and strained through a 40-μm sieve (BD Falcon). Cells were plated in ultra-low attachment plates (Corning) at 1 × 103 cells per well and grown in a serum-free DMEM/F12 phenol-free medium (Corning) containing 1% methylcellulose (Sigma Aldrich), 1% B27 proprietary supplement (Invitrogen), 1% penicillin-streptomycin, 20 ng/ml EGF (Sigma Aldrich), 20 ng/ml basic-FGF (Gibco), and 10 μg/ml heparin (Sigma Aldrich). After 5–7 days, generate secondary tumorspheres were generated; primary spheres were collected and dissociated enzymatically in 0.25% trypsin-EDTA. Single cells were plated as described in conditioned media, which consisted of a 1:1 mixture of DMEM/F12 tumorsphere media (as above) and media from cultured parental cells. The tumorspheres were allowed to grow for 5–7 days before manual counting. Data are presented as the average ± SD of two independent measurements.
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