We examined cellular stress through the visualization of ROS formation in amoebae upon exposure to bacteria in two ways: at the growing edge of a plaque and after acute exposure on washed agars plates for 24 hours. AX4 and cadA amoebae were collected at mid-log phase (2 × 106) in HL5, washed, and resuspended in KK2 buffer at 1 × 107 cells/ml. Amoebae were mixed with 400 μl of overnight K. pneumoniae [optical density at 600 nm (OD600) = 2] in SM and plated on to SM agar. After 24 hours, the plates were scraped and washed three times with KK2 buffer. CellROX Deep Red (Thermo Fisher Scientific) was added to a final concentration of 5 μM, incubated for 30 min, harvested, and washed once with KK2 buffer before fluorescence imaging. To quantitate and compare the relative proportion of CellROX-positive cells, a fluorescence signal threshold that produced 1% positive AX4 cells was applied to all of the images. This threshold was chosen because high ROS levels are not typically observed in wild-type amoebae.

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