HCT116 cells were grown in McCoy's medium and HEK293T cells were grown in DMEM, supplemented with 10% FBS and antibiotics. Sf9 cells were cultured in Grace’ Insect Medium (Gibco) containing 10% inactivated FBS. HCT116 cells with conditional CTC1 disruption were described previously (32 (link)). To introduce wild-type or mutant Flag-CTC1, retrovirus was generated by co-transfecting HEK293T cells with pMIEG3 vector encoding wild-type or mutant Flag-CTC1, gag-pol and env. Viral supernatant was used to infect HCT116 CTC1F/F cells and the cells were then selected by flow cytometry for GFP expression.
Protocol detail
Conditional CTC1 Disruption in HCT116 Cells
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Antibiotics
Cells
Ctc1
Cultured medium
Flow cytometry
Hct116 cells
Insect
Retrovirus
Sf9 cells
Vector
Corresponding Organization : University of Cincinnati
Other organizations : University of South Carolina
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Variable analysis
independent variables
- Introducing wild-type or mutant Flag-CTC1 into HCT116 CTC1F/F cells using retrovirus
- GFP expression in HCT116 CTC1F/F cells after viral infection
- HCT116 cells with conditional CTC1 disruption
- HEK293T cells used for retrovirus generation
- McCoy's medium for HCT116 cells
- DMEM supplemented with 10% FBS and antibiotics for HEK293T cells
- Grace' Insect Medium (Gibco) containing 10% inactivated FBS for Sf9 cells
- Positive control: Introducing wild-type Flag-CTC1 into HCT116 CTC1F/F cells
- Negative control: Not explicitly mentioned
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