Mammalian cells were plated in T-225 tissue culture flasks (BD Falcon, Bedford, MA) and grown up to 90% or higher confluence. When cells reached desired confluence, the monolayer was washed with PBS and the cells were lifted off the plastic culture flask with 5mM EDTA in PBS. This was done to allow access of the bacteria to endothelial cell surface receptors that are normally involved in attachment to the substratum, i.e. receptors that the bacteria may encounter when penetrating the vasculature. In addition, this approach minimizes degradation of mRNA that occurs during harvesting of adherent cells. After lifting, cells were spun for 10 minutes at 1,000 rpm, resuspended in the cell culture medium without antibiotics, and enumerated using a hemocytometer counting chamber. 2610 7 cells per sample were incubated in suspension with either L. biflexa serovar Patoc or L. interrogans serovar Canicola, or without any bacteria, for 1 h and 3 h at room temperature in the cell medium without antibiotics. The MOI (multiplicity of infection) used was 10 bacteria per mammalian cell. After incubation, cells were washed with phosphate buffered saline (PBS) and harvested for RNA isolation. The RNA was purified using RNeasy kit (Qiagen, Valencia, CA) with DNase digestion according to manufacturer's manual. The quality of RNA was checked using a Bioanalyzer (Agilent, Santa Clara, CA).