Epithelium-associated CFU enumeration Association of viable lactobacilli with epithelial cells was assessed by CFU counts as described in detail elsewhere [20]. In brief, at the end of each time period, the cultures were washed twice with CX-5461 in vitro ice-cold PBS and hypotonically lysed for 15 min
in ice-cold HyPure water (Fisher Scientific), followed by adjustment of osmolarity with 2× concentrated PBS (Invitrogen). Serial dilutions were prepared in PBS and 30 μl of each dilution was inoculated on Brucella-based agar plates (PML Microbiologicals). The plates were incubated in an anaerobic chamber (Coy Laboratory Products Inc) containing an atmosphere of 10% hydrogen, 10% carbon GSK872 dioxide and 80% nitrogen at 37°C for 24 h-48 h (until visible colonies were formed), followed by CFU counting. CFU per cm2 epithelial surface area were calculated. NF-κB activation luciferase reporter assay Endocervial epithelial cells stably transfected with pHTS-NF-κB firefly luciferase reporter vector (Biomyx Technology, San Diego, CA)
as described [34] were grown in 96-well plates in hygromycin selection medium until confluence and then colonized with L. jensenii strains as described above. After 24 h, supernatants were collected, cells were lysed with GloLysis buffer and luciferase activity was determined using the Bright-Glo Luciferase Assay System by manufacturer’s protocol (Promega, Madison, WI). Caspase-3 assay Vaginal epithelial cells (Vk2/E6E7) were treated with bacteria, MALP-2 (50 nM) and the proapoptotic agent staurosporine (1 μM) to serve as a positive control. At the end of each incubation period, the epithelial monolayers were lysed in Tris lysis buffer containing protease inhibitor cocktail provided by Mesoscale Discovery (MSD), Gaithersburg, MD, per manufacturer’s protocol. Levels of cleaved and total caspase-3 were measured Selleck Neratinib simultaneously in each cell lysates using an MSD electrochemiluminescence (ECL) mutliplex assay and Sector Imager 2400 with Workbench software (MSD).
Soluble immune mediators assays Concentrations of interleukin (IL-1α, IL-1β, IL-6, TNF-α, IL-8, RANTES, MIP-3α, and ICAM-1) were measured in cell culture supernatants simultaneously using an MSD multiplex assay, Sector Imager 2400, and Workbench software. Levels of IL-1 receptor antagonist (IL-1RA) and the antimicrobial peptide secretory leukocyte protease inhibitor (SLPI) were measured by Quantikine ELISA (R&D Systems, Minneapolis, MN) using a Victor2 reader (Perkin Elmer Life Sciences, Boston, MA). mCV-N detection and functional recovery Cell culture supernatants collected from the vaginal and cervical colonization models were sterilized CB-839 in vivo through 0.2 micron PharmAssure’s Low protein binding syringe filters with HT Tuffryn Membrane (Pall Corporation, Port Washington, NY).
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