Hi fbs

Vero cells (African green monkey kidney cells, ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Waltham, MA) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS, HyClone, Marlborough, MA) at 37°C in a 5% CO₂ atmosphere. HMEC-1 cells (human dermal microvascular endothelial cells, ATCC) were cultured in MCDB 131 medium (Gibco) supplemented with 10 ng∙ml⁻¹ epidermal growth factor (Gibco), 1 μg∙ml⁻¹ hydrocortisone (Sigma, St. Louis, MO), 10 mM glutamine (Corning, Corning, NY) and 10% HI-FBS at 37°C in a 5% CO₂ atmosphere. Stocks of Rickettsia conorii strain Malish 7 (ATCC VR-613) and R. amblyommatis strain GAT-30V (CDC, Dr. Chris Paddock) were generated by growing rickettsiae in Vero cells (5% HI-FBS, DMEM) at 34°C in a 5% CO₂ atmosphere. Rickettsiae were purified from Vero cells by differential centrifugation through 33% MD-76R solution (816 mM meglumine diatrizoate, 157 mM sodium diatrizoate hydrate, 1 mM NaH₂PO₄, pH 7.0; 21 000 × g, 4°C, 20 min) and stored at –80°C in SPG buffer (218 mM sucrose, 3.8 mM KH₂PO₄, 7.2 mM K₂HPO₄, 4.9 mM L-glutamate, pH 7.2).

Human bone osteosarcoma epithelial cells (U2OS cells; ATCC) were maintained between 10% and 90% confluence at 37 °C with 5% CO₂ in DMEM (Gibco) with the addition of 10% heat-inactivated fetal bovine serum (HI-FBS) (Corning), 1% penicillin–streptomycin (Gibco) and 1% sodium pyruvate (Gibco), in glass-bottom 24-well plates pretreated with 75 μl diluted Matrigel (250 μl Matrigel (Corning) diluted in 12 ml DMEM) per well at 37 °C for 30–60 min. The DNA plasmid was transiently transfected into U2OS cells using the TransIT-X2 Dynamic Delivery System kit (Mirus Bio) according to the manufacturer’s protocol.