Smolke:Protocols/Adherent cell care and feeding

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Describe the procedures you use when working with adherent cells such as HEK. After everyone has posted contribution we will combine them to create a standard protocol.


  • General comments:
    • I feed or subculture my cells every 48-72 hours.
    • I monitor the density of my cells using the light microscope and estimate the percentage of the dish surface the cells are covering. If the cells are at 60% or lower I will change the media; 70% or higher I will subculture.
    • Whenever I aspirate I use the glass Pasteur pipet sheathed with a clean L1000 pipet tip.
  • Changing media - I aspirate out the media and add more media using a Pipet-Aid. I find that if I add the media too quickly the force of the liquid will dislodge the cells. This is undesirable.
  • Subculturing - I aspirate out the media and add an amount of trypsin equal to about 1/2 the volume of the dish. As with changing media, I add slowly enough to avoid dislodging the cells. I then aspirate out the trypsin. This step removes the cells that adhere to the dish weakly or not at all. I add more trypsin, this time an amount equal to about 1/3 the volume of the dish. Dislodging the cells is no longer a concern. I return the dish to the incubator for about 5 minutes or until I can see the cells floating in the liquid rather than attached to the dish. I add an amount of media equal to about 2/3 the volume of the dish and pipet almost the whole volume up and down with the Pipet-Aid. This step breaks up any remaining clumps of cells and disperses them into a homogeneous suspension.
    • If I am simply maintaining the cell line I may not count the cells. I add 40-100μL of the trypsinized cell mixture to a new dish of media. This method has generally worked for me. However, if the resulting cell density is too low (10% or lower) the cells will take a long time to repopulate the plate (up to a week) and even when they do they grow in dense colonies rather than evenly throughout the plate. This is undesirable.
    • If I am preparing for a transfection I will count the cells at this point, which allows me to calculate the concentration of the trypsinized cell mixture. After pipetting up and down with the Pipet-Aid again (the cells settle out of suspension in the time it takes to count using the hemocytometer) I dilute the cell mixture with media in a new Falcon tube to reach the concentration I want for the transfection, then aliquot the resulting mixture into the wells of a multi-well plate.


  • General comments:
  • Changing media -
  • Subculturing -


  • General comments: comments here refer specifically to HEK cells.
    • Volumes
      • 24-well plate: 0.5 ml/well
      • 6-cm dish: 3 ml total
      • 10-cm dish: 10 ml total
      • I was taught to use these volumes by Steph/Chase. However, please note that they don't actually scale with the growth areas of the dishes (2 cm^2 for 24-well plate, 21 cm^2 for 6-cm dish, and 58 cm^2 for 10-cm dish). Therefore, plates/dishes seeded at the same density in terms of million cells/ml will not have the same confluency.
    • General techniques
      • Lift lid and tilt plate with one hand while aspirating/pipetting with the other
      • The lid should cover the plate as much as possible, providing just enough opening to perform the required task
      • The lid opening should face away from the hood opening (i.e., away from you and toward the back wall)
      • Tilt the plate toward the back when aspirating
      • Eject media gently against the side wall of the dish to avoid dislodging cells
      • When aspirating, immerse the pipet tip in the liquid but don't touch the plate until the very end to avoid aspirating away cells
    • As with all cell culturing, it is desirable to be as consistent as possible in protocol and timing. Therefore, I always count the cells when I subculture and seed at the same density each time (unless I have specific reasons to seed at a different density). I also feed the cells on a regular schedule.
  • Changing media
    • Aspirate away old media and add fresh media as described above; add antibiotics (for selection pressure) as necessary
    • If seeding 6-cm dish at 0.02 million cells/ml (see below), feed cells 3 and 5 days after seeding; cells should be ready for subculturing on day 6
  • Subculturing
    • Aspirate away old media
    • Wash plate with trypsin (800 ul for 6-cm dish; 3 ml for 10-cm dish) and quickly aspirate away
    • Add another volume of trypsin (same as above) and store in 37C incubator for 10 min
    • Neutralize with media (1 ml for 6-cm dish; 3 ml for 10-cm dish)
    • Mix well by pipetting
    • Sample 10 ul and place in 600-ul microfuge tube
    • Dilute with 90 ul media (make sure it's well mixed)
    • Add 12.5 ul diluted cells to 12.5 ul Trypan Blue
    • Count cells with hemocytometer: count 4 quadrants and divide by 20 to get million cells/ml
    • Seed new dish of the appropriate size at 0.02 million cells/ml (make sure cells in the original dish are well resuspended before you take an aliquot to seed the new dish)


  • General comments:
  • Changing media -
  • Subculturing -


  • General comments:
  • Changing media -
  • Subculturing -