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Microneme secretion assay

CS Catherine Suarez
GL Gaëlle Lentini
RR Raghavendran Ramaswamy
MM Marjorie Maynadier
EA Eleonora Aquilini
LB Laurence Berry-Sterkers
MC Michael Cipriano
AC Allan L. Chen
PB Peter Bradley
BS Boris Striepen
MB Martin J. Boulanger
ML Maryse Lebrun
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Freshly egressed T. gondii tachyzoites (TATi_TgRASP2-HA3 and KD-RASP2-HA3 pre-treated for 48 h ± ATc) were harvested by centrifugation at 600 g, RT for 10 min and washed twice in intracellular buffer (5 mM NaCl, 142 mM KCl, 1 mM MgCl2, 2 mM EGTA, 5.6 mM glucose and 25 mM HEPES, pH 7.2) prewarmed to 37 °C. Parasites were resuspended in DMEM (supplemented with 2 mM glutamine) ± propranolol 500 µM and incubated 20 min at 37 °C. Parasites were centrifuged at 1000 × g for 5 min, 4 °C. Pellets were washed once in PBS and stored at −20 °C. Supernatants were centrifuged at 2000 × g for 5 min, 4 °C and supernatant was used as ESA (excreted/secreted antigen). Pellets and ESA samples were analysed for microneme (AMA1) and dense granule (GRA3) by Western blot.

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