Induced egress assay

Protocol for Induced Egress Assay

1. Freshly egressed parasites were inoculated on a confluent monolayer of HFFs
2. For egress assay with ATc (anhrydrotetracycline)
   • Parasites were treated for 24 hrs prior to egress with or without ATc (1 μg/ml)
   • Freshly egressed parasites from this culture were inoculated on coverslips with HFF monolayer and grown for 30 hr +/−ATc at 37°C. 
3. For egress assay with 49c
   • Parasites were pre-treated with DMSO/49c (1 μM) for 12 hr prior to egress
   • Freshly egressed tachyzoites were plated to a new monolayer of HFF and grown for 30 hr and treated with DMSO or 49c (1 μM) for varying time points. In some cases 49c is washed away 3 hr or 6 hr before induced egress.
4. Following a serum-free DMEM wash, the infected HFF monolayers were incubated with 3 μM of the Ca2+ ionophore A23187 (from Streptomyces chartreusensis, Calbiochem) in serum-free DMEM for 7 min at 37°C. 
5. Cells were fixed with PFA/Glu, and processed for IFA with anti-GAP45 antibody. 
6. 100 vacuoles were counted per strain and scored as egressed or non-egressed. 
7. Results are mean ±standard deviation of three independent biological replicate experiments. 

For live video microscopy of induced egress, parasites were grown on 5 mm Fluorodishes (World Precision Instruments) seeded with HFF monolayers for ∼30 hr at 37°C and egress was induced as described above.