In vitro Tachyzoite to Bradyzoite switch

(adapted from Fux et al, I&I, May 2007, p. 2580–2590 via Kerry Buchholz)

Protocol

  1. Infect confluent monolayers of HFFs with a low MOI (<0.5-5, depending on the strain) of tachyzoites in normal CDMEM Tachyzoites should be syringe released from nice, full, happy vacuoles.
  2. 4 hpi: wash 2x with 3 mL PBS.
  3. Add prewarmed switch media
  4. Place infected flask in 37°C incubator with ambient CO2 (i.e. not supplemented with 5% CO2 as is a standard incubator)

Tips

  • Use very confluent or ‘old’ HFFs (more than a week or two confluent). The parasites seem to switch better in the old cells and the host cells don’t like the media and will round up a bit. Slightly sub-confluent or just-confluent monolayers will look much more sparse after the switch.
  • Make sure all media, especially the switch media, is warm!
  • Letting the tachyzoite infection go 3-4 hours works fine. >4 hours and your switch efficiency drops off.
  • The lower the MOI, the better the switch. The ‘sweet spot’ (between having any parasites and switch efficiency) will vary depending on the strain used.
  • For longer (>4 days) switches it helps to change the media periodically, though the longer you go the more likely you are that host monolayer will die and/or residual Tachyzoites will lyse out. So if you want to do 8+ days make sure you start with a very low MOI and change the media.
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