4 General Cell Culture Methods
41
Procedure
1. Prepare 18- and 36-h-old primary lymphoid cultures in a Primaria 6well plate.
2. Remove the tissue explants.
3. Rinse the substrate attached cells with serum-free medium D.
4. Transfect the cells with naked or Lipofectin (Gibco BRL)-coated pSV3 neo DNA according to the manufacturer's instructions.
5. After transfection, wash each well with serum-free medium, and replace with medium D containing 400 mg/ml Geneticin 418.
Results
According to Tapay et al. (1995), 18-h-old fibroblast-like cell culture began to detach from the surface of the culture flask at 5 days post-transfection, and foci of morphologically distinct round cells appeared. By 79 days post -transfection, these cell foci gave rise to loosely adhesive grapelike arrangements of aggregated cells. These transformed cells have undergone more than 50 passages. In contrast, the Oka cells were not successfully transfected.
Subprotocol 7
Cryopreservation of Cells
Cultured cells can be stored frozen in an ultra low-temperature freezer
or in a liquid nitrogen freezer.
Materials
- Ultra low-temperature freezer
- Liquid nitrogen freezer
- Tank for liquid nitrogen
- Centrifuge and centrifuge tubes
- Programmable freezer
- Cryotubes
- Cultured cells to be preserved
- Liquid nitrogen
- Glycerol
- Dimethylsulfoxide (DMSO)
Equipment
Cells
Chemicals
41
Procedure
1. Prepare 18- and 36-h-old primary lymphoid cultures in a Primaria 6well plate.
2. Remove the tissue explants.
3. Rinse the substrate attached cells with serum-free medium D.
4. Transfect the cells with naked or Lipofectin (Gibco BRL)-coated pSV3 neo DNA according to the manufacturer's instructions.
5. After transfection, wash each well with serum-free medium, and replace with medium D containing 400 mg/ml Geneticin 418.
Results
According to Tapay et al. (1995), 18-h-old fibroblast-like cell culture began to detach from the surface of the culture flask at 5 days post-transfection, and foci of morphologically distinct round cells appeared. By 79 days post -transfection, these cell foci gave rise to loosely adhesive grapelike arrangements of aggregated cells. These transformed cells have undergone more than 50 passages. In contrast, the Oka cells were not successfully transfected.
Subprotocol 7
Cryopreservation of Cells
Cultured cells can be stored frozen in an ultra low-temperature freezer
or in a liquid nitrogen freezer.
Materials
- Ultra low-temperature freezer
- Liquid nitrogen freezer
- Tank for liquid nitrogen
- Centrifuge and centrifuge tubes
- Programmable freezer
- Cryotubes
- Cultured cells to be preserved
- Liquid nitrogen
- Glycerol
- Dimethylsulfoxide (DMSO)
Equipment
Cells
Chemicals