Synchronous Invasion
Required Materials:
25mL Endo buffer at RT
5mL IM at 37C
5mL 1X Fix at 37C
PBS
1T25 infected & >90% lysed HFFs
1 8-well chamber slide with confluent HFFs
Isolate parasites from >90% lysed T25, Syringe 3X each with 20 & 23 Gauge needle,
Pass through filter and rinse with 10 mL Endo Buffer.
Take 8.5 uL for hemocytometer count
Spin at 1000g for 10min
Resuspent in Endo Buffer to 5e6 parasites/mL
Remove all D10 from chamberslide and rinse once with Endo Buffer
Add 200uL(1e6) of parasites in Endo per well
Spin in 96-well plate rotor for 2min at 500g without break (be sure to use another chamberslide as balance)
Place
in 37C water bath so that wells are 50% submerged on the outside wall.
( I use a magnetic stir bar to keep it from floating)
Remove Endo with Pipettor and save for counting.
Add 500uL of IM carefully and start timer.
After desired time, pipette IM in well up and down 3X and discard.
Add 500uL of 1X Fix to well.
Remove Fix after 10 min and replace with PBS.
Repeat for all wells and desired times.
Remove wells and wash slide twice in PBS for 5 min.
Count removed parasites in the saved Endo buffer.
Perform Red/Green stain on the slide.
Buffer Recipies:
IM:
4.75ml D10
0.15ml FBS
0.10ml HEPES
Fix:
2ml 10% Formaldehyde (Histology grade or higher)
4ul 27% Gluteraldehyde
0.5ml 10x PBS
2.5ml ddH2O
Endo Buffer:
44.7 mM K2SO4
10 mM MgSO4
106 mM Sucrose
5 mM Glucose
20 mM Tris-H2SO4
3.5 mg/mL BSA
pH to 8.2