The Protocol for Cell Culture Passaging

Preparation of Cell Growth Media

  1. All reagents should be thawed and exposure to room temperature conditions minimized.

The following reagents should be combined under aseptic conditions in the designated quantities:

  1. DMEM 450 mL
  2. 10% Fetal Bovine Serum (FBS)
  3. 2 mM Glutamine 5 mL
  4. 100 μL Penicillin/Streptomycin
  1. The Glutamine and Pen/Strep solutions should both be thoroughly mixed before their addition.

The OHAUS Analog Mini Vortex Mixer (30392115) can be used for this.

  1. Use an inversion or serological pipette to mix the solution gently. Make sure the mixture remains at 4 °C until use. Before treating cells, warm the media to 37 °C. The OHAUS Digital Dry Block Heater (30392080) and Sand Block Attachment (30400174) can be used to do this. It is also possible to use a water bath, but this will increase the risk of contamination.

Initial Plating of Mammalian Cell Cultures

  1. Use a biological or commercially available source to obtain mammalian cells.
  2. Establish whether the cells are adherent or rely on suspension.
  3. Frozen cells should be thawed quickly and exposure to room temperature conditions minimized.
  4. Add the required amount of prepared media to prepare the sample vessel. A 50 mL culture flask will most likely suffice for both adherent and suspension cells.
  5. The entirety of the initial culture should be pipetted into an appropriate vessel using the recommended cell density. The culture should be gently swirled to make sure that the cells are distributed evenly.
  6. The prepared culture should be immediately placed into an incubator at 37 °C with 5% humidity.
  7. The cells should be checked daily to make sure that they are healthy. At the initial plating, some cell death should be seen.

Splitting of Cells (Without the Use of Trypsin or EDTA)

  1. The initial culture should be allowed to become 80% confluent.
  2. The ratio at which the cells need to be split should be determined, which will often depend on when the cells will be used. The guide below can be referenced for a recommendation.
    1. For an experiment in 1 to 2 days - 1:2 split should be 70-80% confluent.
    2. For an experiment in 2 to 4 days - 1:5 split should be 70-80% confluent.
    3. For an experiment in 4 to 6 days - 1:10 split should be 70-80% confluent.
  3. The cells should be removed from the incubator and placed into an aseptic environment.
  4. For adherent cells, the initial media should be gently removed from the flask using either a pipette or a gentle pour.
  5. The cells should be washed with warm phosphate-buffered saline (PBS). This is done by directly adding, and then quickly removed, a thin layer onto the cells.
  6. The PBS should be completely removed and fresh, warm media added onto the cell culture at the initial volume.
  7. The new flask to be used for the split should be prepared. Media volume should be left out to allow for the addition of new cells. For example, 9 mL of media should be added where a 1–10 split is required. This will allow for the later addition of 1 mL of cell-containing media. The flask should be labeled with the date and passage number. For example, the passage number will be 2 if it is the first time splitting the cells.
  8. A cell scraper should be used to gently scrape the monolayer, which will release the adherent cells into the media. Before moving on to the next step make sure that all cells are detached.
  9. When all cells are suspended, the required volume for the split should be removed and immediately placed into the freshly prepared flask. The flask should then be gently swirled and placed into an incubator at 37 °C with 5% humidity.
  10. Steps 4, 5, 7 and 8 should be skipped for suspension cells. The required amount of cells should be removed directly from the initial culture and placed into a 15 mL tube.
  11. The tube should be centrifuged at room temperature at 1500 rpm for 3 minutes using the OHAUS FC5714 Multi Pro Centrifuge (30314811) with a Swing Out Rotor (30314822 + 30314850)
  12. Once the cells have formed a pellet at the bottom of the tube, the supernatant should be gently removed.
  13. The cells should then be re-suspended in fresh, warm media using a pipette and placed directly into the freshly prepared flask. This should be gently swirled and placed into an incubator at 37 °C with 5% humidity.
  14. If there are any cells left over, these can either be disposed of or further passaged to create additional sub-cultures.

OHAUS Products Used Within this Procedure

Analog Mini Vortex Mixer

Analog Mini Vortex Mixer

Digital Dry Block Heater

Digital Dry Block Heater

Sand Block Attachment

Sand Block Attachment

FC5714 Multi Pro Centrifuge

FC5714 Multi Pro Centrifuge

Swing Out Rotor

Swing Out Rotor

About OHAUS Corporation

OHAUS Corporation manufactures an extensive line of weighing products, laboratory equipment and analytical instruments that meet the weighing and measurement needs of virtually every industry.

They are a global leader in the laboratory, industrial and education channels, as well as a host of specialty markets, including the food preparation, pharmacy and jewelry industries. An ISO 9001:2008 manufacturer, OHAUS products are precise, reliable and affordable, and backed by industry-leading customer support.

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Last updated: Nov 5, 2019 at 7:41 AM


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