). The TER for polycarbonate filters without cells was approximately 100 Ω/cm2. The upper chamber of the transwell apparatus was inoculated with leptospires at a multiplicity of infection (MOI) of 100 by adding 500 μL of bacteria which were resuspended in 1:2 v/v ratio of DMEM and EMJH media. Duplicate transwell chamber assays were performed for each leptospiral strain which were tested. Aliquots were removed from lower chamber (100 μl) at 30, 120 and 240 min and the number of leptospires was counted in triplicate by using the Petroff-Hausser chamber. The ability of leptospires to translocate
MDCK polarized monolayers was determined by calculating the proportion of leptospires in the lower chamber in comparison to the initial inoculum for duplicate assays at each time point. The ANOVA test was used to determine significant differences in the proportions of translocating leptospires and TER values Citarinostat molecular weight selleck chemicals llc obtained
during incubations with different leptospiral strains. ELISA for binding to extracellular matrix components The adhesion of live L. biflexa strains to immobilized fibronectin was measured with an ELISA. Two to three × 108 cells in serum-free EMJH or medium alone was incubated at 30°C for 1 h in a microtiter well pre-coated with 1 μg of fibronectin (from human plasma or foreskin fibroblasts, Sigma-Aldrich), collagen type I (bovine skin, Sigma-Aldrich), collagen type IV (human placenta, Sigma-Aldrich), laminin (murine, Sigma-Aldrich), elastin (human skin, Elastin Products Company, Owensville, MO), or left
in PBS, pH7.2, overnight at 4°C. Uncoated sites in the well were covered with Protein-Free Blocker (Thermo Scientific) before the addition of cells. Adherent cells were fixed with 4% formaldehyde (Thermo Scientific) at room temperature for 1 h, tagged with a rabbit polyclonal antibody for intact L. biflexa (MyBioSource), and detected by spectrometry at 450 nm to PRKD3 measure the activity of horseradish peroxidase conjugated to a donkey antibody for rabbit IgG (GE Healthcare). Backgrounds from uncoated wells (PBS) and medium only were subtracted. Triplicate assays were done and statistically significant differences in adhesion were determined with one-way ANOVA compared to the wild-type cells. Acknowledgements This work was supported by the Institut Pasteur, Paris, France; the French Ministry of Research ANR-08-MIE-018, the Fiocruz-Pasteur Scientific Cooperation Agreement, Oswaldo Cruz Foundation (PDTIS RVR05), Brazilian National Research Council (INCTV), VA Medical Research Funds, and the National Institutes of Health (grants D43 TW00919, R01 AI34431 and U01 AI088752). This research was conducted by C.P. Figueira in partial fulfillment of the requirements for a Ph.D. from Goncalo Moniz Research Center, Oswaldo Cruz Foundation, Brazil. Electronic supplementary material Additional file 1: surface immunofluorescence assays in L. interrogans. Immunofluorescence assays were performed with L.
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