Facscalibur laser cytometer

For in vitro rickettsial infection, macrophages were infected with R. australis at a multiplicity of infection (MOI) of 10:1, 6:1, 2:1 or as indicated. To synchronize bacterial internalization, rickettsiae were centrifuged onto the cells at 560 × g for 5 min. Cells were incubated continuously at 37°C in 5% CO₂. Uninfected cells served as negative controls. Positive controls were the cells primed with LPS (200 ng/ml) for 4 hours followed by incubation with ATP (5 mM) for 45 min. The viability of infected mouse BMMs was examined by trypan blue staining and LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit (Life Technologies, Grand Island, NY) in accordance with the manufacturer’s protocol. Positive control was composed of half of them alive and half of them dead. Flow cytometry was performed on 30,000 cells with a FACSCalibur laser cytometer (Becton-Dickinson, BD Biosciences, San Jose, CA). Data analysis on the entire ungated cell population was performed using FlowJo software.

HMEC-1 cells were washed with PBS before the addition of 0.025% (wt/vol) trypsin (Gibco, Grand Island, NY) to detach and lift cells from the culture surface. Fresh medium was added to the suspension to inactivate trypsin, and the cells were centrifuged at 330 × g for 10 min at 4°C. The pellet was washed with precooled PBS. Cells were first stained using a fixable LIVE/DEAD Near-IR Dead Cell Stain Kit (Life Technologies, Grand Island, NY) in accordance with the manufacturer’s protocol. Annexin staining solution was prepared using the manufactures provided 10× Annexin V binding buffer (10%), annexin V-PE (2%), and water (88%), and incubated with 1×10⁶ cells for 15 min at room temperature in the dark. Cells were washed in binding buffer, fixed with 1% paraformaldehyde containing binding buffer, and stored at 4°C until subjected to fluorescence-activated cell sorting analysis. Flow cytometry was performed on ⩾10,000 cells with a FACSCalibur laser cytometer (Becton-Dickinson, BD Biosciences, San Jose, CA), and data analysis was performed using FlowJo software.