This question is part of the following Ask The Expert session:
The best time to perform transfection after thawing depends on the cell type. A wide variety of immortalized cell lines have optimal performance after passage 4-5 and until passage 20-25. If transfection is performed on low passage cells, post thawing, lower transfection performance will be observed; while cells that are maintained for higher passage numbers, will have an inaccurate and misleading high transfection efficiency. We are also aware of one cell line, NIH 3T3, that has better transfection at a higher passage number, from 10-40. There are also cell lines, such as primary endothelial cells, primary smooth muscle cells, primary corneal epithelial cells that have a much smaller passage window for optimal transfection performance. Since passage number can affect the growth rate of the cells, which can directly affect transfection, we suggest to test the range of recommended reagent doses, which can be found in the revised protocols on our website.