Lionheart fx microscopy

For the mPTP assay, HT-29 cells were seeded at 2.0 × 10⁵ cells/well into 24-well plates overnight in complete media. Cells were infected at an MOI of 1 and incubated at 37 °C with 5% CO₂. After infection, the cells were washed with HBSS 1X, the medium was replaced with fresh complete Fluorobrite DMEM medium (Thermo Fisher Scientific, Waltham, MA, USA, A1896701), and an Image-iT LIVE Mitochondrial Transition Pore Assay Kit (Thermo Fisher Scientific, I35103) was used to acquire fluorescence images with Lionheart-FX microscopy (Agilent BioTek, Santa Clara, CA, USA) at different time points of infection. This kit utilizes 1 µM of Calcein-AM (green), which accumulates in the mitochondria of live cells, and 1 mM of Cobalt (Co²⁺) to quench the signal of Calcein when the mPTP is open for a prolonged period. As an experimental control, we use 0.5 µM of ionomycin, an ionophore that induces the opening of the mitochondrial pore. The kit was employed according to the manufacturer’s specifications, and cells were fixed with 4% formaldehyde (Thermo Fisher Scientific, 28908) and the samples were mounted using Prolong Diamond Antifade Mountant (Thermo Fisher Scientific, P36961).

For the mitochondrial fragmentation assay, HT-29 cells were seeded at 2.0 × 10⁵ cells/well into 24-well plates overnight in complete media. Cells were infected at an MOI of 1 and incubated at 37 °C with 5% CO₂. After the infection, the cells were washed with HBSS 1X, the medium was replaced with fresh complete Fluorobrite DMEM medium (Thermo Fisher Scientific, A1896701), a 0.2 µM Mito Tracker Red CMXRos (Thermo Fisher Scientific, M7512) was used for 15 min, the cells were fixed with 4% formaldehyde (Thermo Fisher Scientific, 28908), and the samples were mounted using Prolong Diamond Antifade Mountant (Thermo Fisher, P36961) to visualize HT-29 mitochondria morphology with Lionheart-FX microscopy (Agilent BioTek) at different time points of infection.