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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

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