Maintaining Dictyostelium axenic cell lines

Janet Smith | Boston Biomedical Research Institute

Maintaining cells in shaking cultures

Ax3 doubling time is 8-12 hours. Cultures will typically need to be split back every 3-4 days.


  1. Place 10 ml HL-5 in a 50 ml flask.

  2. Inoculate with 100 μl of a culture in mid- to late-log late–log phase (5x106/ml).

  3. Shake cells at 200 rpm at 21°C.

  4. Split cells back before they get to 5x106/ml.

Maintaining cells on plates

Plates will typically need to be split back every 3-4 days.

  1. Place 10 ml HL-5 in a 100 mm plate.

  2. Inoculate with 100 μl that was removed from a nearly confluent plate by dragging the pipet tip along the bottom of the plate while sucking up the liquid.

  3. Place at 21°C.

  4. Split cells back when the plate is ∼90% confluent.

Developing cells on K. aerogenes

Cells should not be passed for more than three weeks. After that time, you can develop them and restart cultures from the resulting fruiting bodies, or start new cultures from frozen spore stocks.

  1. Make a lawn of K. aerogenes by spreading 200 μl of an O/N culture onto a 100 mm SM/5 plate. Add about five sterile glass beads and gently roll them around on the surface of the plate to evenly spread the lawn.

  2. Allow to grow O/N at 37°C.

  3. Spot about 10 μl Dictyostelium cells onto the plate and incubate at 21°C.

  4. Fruiting bodies will appear in 3-4 days. Collect a few of these with a sterile loop and innoculate a plate of HL-5.

Making a frozen spore stock

  1. Grow up 2x108 healthy, log phase, Ax3.
  2. Spin down a 1500 RPM in table-top centrifuge.
  3. Resuspend cells in 25 ml MES Buffer (20mM MES, pH6.4, 2mM MgCl2, 0.2mM CaCl2)
  4. Spin cells again, and resuspend in 4 mls MES Buffer.
  5. Pipette evenly onto MES Agar plate and allow cells to attach 30-45 min.
  6. Carefully remove buffer by aspiration, trying not to lose too many cells.
  7. Allow cells to develop at 20°C for 30-40 hours. You should see fruiting bodies by this point.
  8. Using a sterile loop, collect spores from 1 plate of fruiting bodies into 0.5 ml sterile MES Buffer. The mixture will resemble grated ginger.
  9. Using a wide-bored pipet tip, aliquot 0.1 ml into 5 freezer vials.
  10. Store at -80°C or in liquid N2. To bring out spores, thaw and place in HL-5.