Protocol – Subculturing adherent cells growing in the serum-containing medium using Trypsin-EDTA


  • Trypsin-EDTA method also referred to as trypsinization, is a most commonly used method for passaging/subculturing adherent cells.
  • Cells to be subcultured are first washed with Ca2+-free and Mg2+-free PBS and subsequently treated with Trypsin-EDTA solution.
  • Brief incubation with Trypsin-EDTA disrupts cell-cell and cell-substratum interactions, resulting in the single cell suspension.
  • Trypsin, a proteolytic enzyme, disrupts these interactions by proteolysis and EDTA by chelating divalent cations.
  • Single cell suspension can be used for the determination of cell number and preparation of fresh culture.


  • Reagents and solutions:
    • 1 X Trypsin-EDTA solution (Room Temperature / 37°C)
    • PBS (Ca2+-free and Mg2+-free) (Room Temperature / 37°C)
    • Complete growth medium (Room Temperature / 37°C)
  • Equipment and disposables
    • T25 flask/Tissue culture dishes
    • Pipette and pipette aid (Glass Pipettes)
    • Laminar flow hood
    • Beaker to discard the waste

Starting material: 90% confluent T25 flask/60 mm dish

Prior to start:
  • Place all reagent bottles – Trypsin EDTA, PBS and complete medium in 37°C water bath.
  • Wipe laminar flow hood with 70% ethanol, turn on UV light for 20 – 30 min. After 20 min, turn off the UV light and start the air flow. Let the air flow for 10 min before use.
  • Now wipe the surface of bottles of all reagents and place them in the laminar flow hood.
  • Properly label all dishes/flask with the date of subculture, passage number, cell name and your name.
  • Check cells under the microscope to make sure cells are healthy and are not contaminated.


Subculturing of subconfluent (90% confluent) culture growing in a T25 flask/60 mm culture dish

  • Do all operations aseptically.
  • All transfer of medium should be done inside the laminar flow hood.
  • Wear lab coat and disposable latex gloves at all times.
  • Do all steps which involve changing medium from cells quickly to avoid any risk of drying of cells.
  • Don’t decant medium from the flask/dish. This can increase the risk of contamination.


Step 1: Remove and discard culture medium from the flask and wash cells with PBS free of Ca2+ and Mg2+.
  • Take out the flask from the incubator and place inside laminar flow hood.
  • Slightly tilt the flask/dish. All medium will be collected at tilted side. Now remove and discard all medium.
  • Add 3 – 4 ml PBS. Swirl or tilt the dish/flask in opposite direction 2-3 times. Remove and discard PBS.
  • Serum in the culture medium has trypsin inactivating activity.
  • PBS washing will remove dead cells, cell debris and remaining growth medium.
  • To remove liquid from the flask/dish, you can use vacuum aspirator. Vacuum aspirator is very quick and convenient way to remove liquid from the dish. If you don’t have Vacuum aspirator, use pipette and pipette aid to remove liquids from the flask.
  • Always keep the flow of PBS on side walls of the culture vessel. This will minimize the cell detachment due to flow of the liquid.
  • While pipetting PBS into the flask, take care that the flow of PBS should not disturb cell monolayer.
  • Some cell lines are loosely adherent. In such case, care must be taken to avoid any loss while removing culture medium and washing with PBS.
Step 2: Add Trypsin – EDTA solution and incubate 1 – 5 min at 37°C.
  • Add 1 ml Trypsin-EDTA solution and gently spread trypsin solution all over the cell surface by swirling/tilting the vessel . Incubate at 37°C (in incubator) until cells appear rounded and start detaching from the substratum (it can take 1-5 min for most of the cell lines).
  • Gently tap the flask to remove all cells from the surface.
  • 0.5 – 1 ml Trypsin-EDTA solution is sufficient for T25 flask/60 mm dish. Depending on the convenience, one can add more trypsin-EDTA solution. The amount of Trypsin-EDTA should be sufficient to cover all the cell surface of the culture vessel.
  • For each cell line, one should optimize trypsin-EDTA treatment condition (incubation time and concentration). Some cell types are strongly adherent and require a higher concentration of the trypsin-EDTA solution as well as longer incubation time.
  • Examine the cell morphology under the microscope every minute if you don’t have any idea how long trypsin-EDTA treatment is required for your specific cell line.
  • Make sure trypsin – EDTA solution covers all cell surface. Each cell should come in contact with trypsin solution. Incomplete trypsin treatment may result in cell clumps.
  • Right trypsin-EDTA treatment condition is crucial for successful trypsinization process. While overtreatment will cause cell death, undertreatment results in cell clump and undetached cells in the culture dish.
Step 3: Inactivate trypsin by adding fresh serum containing medium
  • Wash out all the cells from the surface by pipetting the fresh culture medium (4 ml) all over the surface.
  • Disperse all cell clumps by pipetting 2 – 3 times.
  • Serum has trypsin inactivating activity. If you are using serum-free medium, inactivate trypsin by adding other trypsin inhibitors e.g., soybean trypsin inhibitor.
  • While transferring the medium in the vessel, keep the flow of medium toward the surface where cells were attached.
  • If there are cell clumps, disperse them by several rounds of pipetting. More pipetting can cause cell death!
Step 4 (optional): Determine cell number
  • Transfer all cell suspension to a centrifuge tube. Collect cells by centrifugation.
  • Resuspend cells an appropriate amount of complete medium (5 ml) and determine cell number using haemocytometer or any other method.
  • Cell counting is not necessary for the regular maintenance of cell culture. Most researchers use split ratio to seed cells in a fresh culture dish.
  • Split ratio is a rough estimation of how many culture dishes can be prepared from the existing cell culture. For e.g., you can prepare 5 to 10 flask/dish from the cell culture (90% confluent) if the split ratio is 1:5 to 1:10.
Step 5: Prepare fresh culture dish from the cell suspension
  • Transfer an aliquot of cells into fresh culture flask/dish.
  • Add fresh growth medium. Pipette cells to resuspend cells again. Wright all details including your name, cell line name, passage number and subculture date.
  • Discard remaining cell suspension if you don’t need them.
  • Keep the flask in the incubator. Open the lid of the flask slightly for air exchange if you are using sodium bicarbonate containing culture medium. Tighten the lid if you are using vented cap flask.
  • Use recommended seeding density or split ratio to decide how many cells should be transferred to fresh dish/flask. Generally 1:5 – 1:10 split ratio works well for fast-growing cell lines.
  • If you need to make many culture dishes/flasks, prepare master mix before transferring suspension to the fresh flask.