Evos automated microscope

To measure growth of C. burnetii in wild-type and acat-1^-/- MH-S cells, 5x10⁴ cells/well were infected for 1 hour in a 48 well plate, washed with PBS, and then incubated with media containing respective vehicle and inhibitors. At the indicated time points, the media was removed and cells were incubated with sterile water for 5 min, pipetted up and down and the lysate diluted 1:5 in 2% FBS-RPMI. Serial dilutions were added to 24 well plate containing confluent monolayers of Vero cells, incubated for 5 days, fixed with methanol and stained with rabbit anti-C. burnetii antibody as well as DAPI to confirm monolayer integrity. Four fields per well were captured on an Evos automated microscope (ThermoFisher) with 4X objective and fluorescent foci units were quantitated using ImageJ. Each independent experiment was performed in duplicate.

hiPS-MΦ were resuspended in macrophage media supplemented with 20 µL/mL ReadyProbes Cell Viability Kit, Blue/Green (ThermoFisher-R37609). hiPSC-MΦ were then plated at 3 × 10⁴ cells per well in a clear bottom 96-well plate and treated with: 10 ng/mL TNFα (Peprotech) 10 or 100 ng/mL LPS (Sigma), 25 µg/mL poly(I:C) (Sigma), 10 µM zVAD-fmk (BD Pharmingen^TM-550377) and/or 3 µM GSK872 (BioVision-2673-5). Cells were imaged every hour for 24 h in an EVOS automated microscope (Thermofisher) in a humidified 37 C, 5% CO₂ incubation chamber. Number of green (dead) and live (blue) nuclei was calculated using imageJ. Percentage of viable cells over time was defined as the ratio between live and dead cells.