Fluoroblokhts 24 well multiwell permeable support system with a 3.0 μm high density polyester pet membrane

Taxis was tested using the Corning FluoroBlokHTS 24-well multiwell permeable support system with a 3.0-μm high density polyester (PET) membrane (Corning, New York, USA) as described in Fig. 2. This filter system was chosen to allow the transfer of bacteria but not of amoeba. The plates were measured using a Synergy HT plate reader (Biotek, USA; used throughout this work).
Pseudomonas aeruginosa and amoeba were cultivated and prepared as described above. Bacteria were diluted to OD₆₀₀ of 0.1, and amoebae were diluted to 1 × 10⁴ cells/mL (both in TBSS). A total of 750 μL of the amoeba suspension was added to the bottom chambers of columns 1 to 3 of the base plate, columns 4 to 6 were loaded with 750 μl of TBSS buffer and used as the control, and the filter system was mounted onto the base plate. The top chamber of a single well was loaded with 100 μl of the bacterial culture and read kinetically for bottom fluorescence every 4 seconds for 2 minutes, using the following reading parameters: excitation, 485 nm/20; emission, 528 nm/20; and gain, 60. The process was repeated for all wells. At the end of the experiment, the filter insert was removed and five wells were sampled (10 μL each from the bulk liquid away from the bottom of the well) and examined microscopically to verify that amoebae did not detach from the bottom of the well.

Taxis was tested using the Corning FluoroBlokHTS 24-well multiwell permeable support system with a 3.0-μm high density polyester (PET) membrane (Corning, New York, USA) as described in Fig. 2. This filter system was chosen to allow the transfer of bacteria but not of amoeba. The plates were measured using a Synergy HT plate reader (Biotek, USA; used throughout this work).
Pseudomonas aeruginosa and amoeba were cultivated and prepared as described above. Bacteria were diluted to OD₆₀₀ of 0.1, and amoebae were diluted to 1 × 10⁴ cells/mL (both in TBSS). A total of 750 μL of the amoeba suspension was added to the bottom chambers of columns 1 to 3 of the base plate, columns 4 to 6 were loaded with 750 μl of TBSS buffer and used as the control, and the filter system was mounted onto the base plate. The top chamber of a single well was loaded with 100 μl of the bacterial culture and read kinetically for bottom fluorescence every 4 seconds for 2 minutes, using the following reading parameters: excitation, 485 nm/20; emission, 528 nm/20; and gain, 60. The process was repeated for all wells. At the end of the experiment, the filter insert was removed and five wells were sampled (10 μL each from the bulk liquid away from the bottom of the well) and examined microscopically to verify that amoebae did not detach from the bottom of the well.