1. At 5-7 d after transfection, G418 resistant cells are split to separate cells and to allow individual colonies to form. The cells are maintained in G418-containing medium until they form large colonies.
2. Thirty to forty colonies are isolated with a pipet under an inverted microscope (see Note 4).
3. The cells of these colonies are placed in 24-well plates and separated by pipetting, each well containing one group of cells. These cells are maintained in the same G418-contain-ing medium until each well contains enough cells to split and to analyze.
4. Normally, cells in one well of 6-well plates would be enough for Western blot analysis and cells in 24 or 12-well plates can be used for immunofluorescence examination.
5. We save stably transfected cells by freezing them in liquid nitrogen. To do so, cells are treated with Trypsin-EDTA medium and then collected by centrifugation. The cells are then resuspended in the freezing medium at 3-5 x 106 cells /mL. The cells are first placed in a -80°C freezer for overnight or a couple of wk. For a long-term storage, the cells are transferred from the -80°C freezer to a liquid nitrogen tank (see Note 5).
Was this article helpful?