Splitting Mammalian Tissue Culture Cells

  • 1. Examine the flask of cells in the TC microscope. Make sure it is not contaminated, that the cells are healthy and that there are sufficient cells to split.
  • 2. Materials needed:
    • Trypsin (do not warm up)
    • PBS – (do not warm up)
    • Medium (warm at 37)
    • Pipettes
    • Flasks
    • Dishes (if splitting for experiments)

  • 3. Protocol:
    • a. Remove medium from flask, discard in waste beaker
    • b. add ~5mls of sterile PBS – to flask, swirl, remove to waste beaker
    • c. add ~0.5 ml of sterile Tryspin. Tilt flask so trypsin covers surface. Tighten cap. Place in incubator, or leave in hood, if you want the process to go more slowly.
    • d. check cells under microscope at 3 mins, and again until they are starting to detach.
    • e. add ~5mls of appropriate medium. Triturate cells AVOID bubbles.
    • f. distribute cells to new flasks and dishes.

    How many cells to put in each flask? If original flask was full, then use ~1/10th to ~1/5th of the volume; use more if you need cells in a day or two, use less if you don't need cells for a few days.

    For dishes: you want a nice monolayer of cells to form within one or two days. I add a drop or two of cells AND examine in the microscope. You can quickly get a feel for the number of rounded cells and how heavy the plating will be. You can measure the exact volume and keep track if that helps. Remember to distribute cells on the coverslip or Mattek surface. Flasks – use 4ml of medium; dishes use 1.5 mls of medium. Do not overfill the dishes.