24 wells microplates

Epithelial cells were harvested from Corning culture flasks with 0.25% trypsin (Sigma-Aldrich, Buchs, Switzerland), washed with fresh medium and seeded, 1 day before infection, at 2-5 × 10 5 cells per well in 24-wells microplates (Corning) or at 4 × 10 6 cells per 25 cm 2 flasks for RNA extraction and total protein preparation. Cells were infected with a 1/2000 dilution of bacteria (MOI 0.1-1). Plates were then centrifuged at 1790 g for 10 min at room temperature. After 15 min of incubation at 37 • C in the presence of 5% CO 2 , cells were washed with fresh medium to remove noninternalized bacteria and were then incubated for different periods of time at 37 • C in a 5% CO 2 atmosphere.

Insect cells were seeded at 1x10 5 cells per well in 24-wells microplates (Corning) the day before infection. Infection was performed at 28°C or 27°C, as described in [24] , with a 5 days-old culture of bacteria in A castellanii diluted, if not otherwise described, 1:1000 for W. chondrophila (MOI: 1-20) and P. acanthamoebae (MOI: 0.1-1) and 1:2000 for E. lausannensis (MOI: 1-10)