▲ CRITICAL STEP We typically passage cells every 2–3 d at a split ratio of 1:4 or 1:8, never allowing cells to reach more than 70% confluency. Cells are discarded upon reaching passage number 15. 8Preparation of cells for transfection. Plate the well-dissociated cells onto 24-well plates in D10 medium without antibiotics 16–24 h before transfection. Seed the cells at a density of 1.3 × 105 cells per well in a total volume of 500 μl. Scale up or down according to the cell line supplier's manual as needed.
