Importance of cell passage number and cell confluency for efficient transfection

The viability, confluency and passage number are all vital parameters for a successful transfection.  Here are helpful hints for each of these aspects:

• Viability
  • Viability is the percentage of living cells in a suspension
  • Determined using trypan blue exclusion
  • Cells are considered to be healthy if viability is greater than 90%
• Confluency
  • Confluency is the percentage of a well surface covered by cells
  • Determined by naked eye by visualization under a microscope
  • Transfection is successful when cells are near 50-80% confluent and actively growing; too few cells results in poor growth due to lack of cell-to-cell contact, and too many cells results in contact inhibition and lack of foreign DNA uptake
• Passage Number
  • Passage number is the number of times a cell culture has been removed from a surface and replated; typically, labs label each cell line they receive as passage 1
  • Common practice is to not use cells after 20 to 30 passages
  • For planning purposes, create ample freezer stocks at early passage numbers to ensure similar passage numbers for repeat experiments