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Cell Passage Protocol

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This protocol describes the entire life cycle of a mammalian cell culture. It starts with seeding the flask and finishes with harvesting, counting, and the preparation of a normalized cell suspension.  It is written for HEK297 but can be adapted for other cells.

Protocol Version 1.1
Active Time 30 min
Process Time 72 hours


Equipment Needed

Supplies Needed

Detailed Instructions

Day 1: Seeding

  1. Affix one of the labels to the T-75 flask. 
  2. Add 13 ml of DMEMS 10% FCS in the T-75 flask standing.
  3. Add 2 ml of HEK293T cells in suspension at 1 million cells/ml.
  4. Close the flask and place it horizontally.
  5. Spread the cells with gentle rotating movements.
  6. Place in the incubator for 48 hours.

Day 3: Harvesting

  1. Confirm cell culture convergence under the microscope. It should be between 50% and 80% convergent. 
  2. Affix preprinted labels to the 15 ml and 50 ml tubes 
  3. Aspirate culture medium with the Pasteur pipette.
  4. Add 2 ml of Trypsin EDTA solution to remove all traces of serum that contains trypsin inhibitor, spread over the culture, and aspirate immediately.
  5. Add 3 mL of trypsin-EDTA solution to the flask. Spread the solution over the entire cell culture and let it rest horizontally. 
  6. Add 8 ml of DMEMS 10% FBS to the 15 ml tube.
  7. After a couple of minutes, take the flask and observe the cells detach from the flask surface with the naked eye by gently rocking the flask. Put the flask vertical and let the cells accumulate at the bottom.
  8. Remove the cells in suspension with a 5 ml pipette and add to the 15 ml conical tube resulting in a total volume of 11 ml.
  9. Count cells and measure their viability. 
  10. Report cell numbers and viability in the cell culture record.
  11. Spin the 15 ml tube produced in Step 8 (1000 RPM / 200g) for three minutes at room at 20C. 
  12. Add a volume V of media to a 50 ml tube with V being the number of cells harvested in millions minus 2 ml.
  13. When the centrifugation is complete, aspirate the supernatant being careful not to aspirate the cells in the pellet. 
  14. Resuspend cells in 2 ml of media.
  15. Transfer 2 ml to the 50 ml tube prepared in Step 12.


  • This protocol should produce 16 to 20 million cells with a viability of 90% or greater. 


  1. This culture can be harvested on Day 4. It is likely to be slightly overgrown 80% to 100% convergence but should still be able to be rescued. 
  2. Seed the culture with 1 million cells instead if you know that you will harvest them on Day 4.