Hras1, H340T and H245T tumor cell lines were establish from thyroid tumors from HrasG12V/Pten−/−/TPO-Cre mice. Thyroid tumors were dissected and minced, followed by digestion in a solution of 1 mg/ml collagenase Type I (Sigma) and dispase (Gibco) in Hank's Balanced Salt Solution at 37°C with gentle shaking for 1.5 hours. Following digestion, samples were centrifuged at 1200 rpm for 3 minutes and resuspended in F12 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 2 mM L-Glutamine (Gibco), and Penicillin/Streptomycin/Fungizone (Sigma). The samples were then plated into tissue culture flasks and maintained at 37°C in 5% CO2. To ensure removal of contaminating stromal cells and purity of the tumor cell lines, all cell lines were genotyped using primers specific for Pten and Pten recombination [24 (link)].
Protocol detail
Establishment of Hras1 Thyroid Tumor Cell Lines
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Cell lines
Collagenase i
Digestion
Dispase
Fungizone
Glutamine
Hank balanced salt solution
Mice
Penicillin
Primers
Pten
Recombination
Streptomycin
Stromal cells
Thyroid tumors
Tissue
Tumor cell lines
Corresponding Organization :
Other organizations : University of Arkansas for Medical Sciences, Winthrop Rockefeller Foundation
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Variable analysis
independent variables
- H340T and H245T tumor cell lines established from thyroid tumors of Hras^G12V/Pten^-/-/TPO-Cre mice
- Purity of the tumor cell lines
- Hras1 tumor cell line established from thyroid tumors of Hras^G12V/Pten^-/-/TPO-Cre mice
- Tissue culture conditions (37°C, 5% CO2)
- Cell culture media (F12 medium with 10% FBS, 2 mM L-Glutamine, Penicillin/Streptomycin/Fungizone)
- Positive control: Primers specific for Pten and Pten recombination to ensure purity of the tumor cell lines
- Negative control: Not explicitly mentioned
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