By this procedure, we isolated epithelial cells that stained positive for the epithelial antigens Ber-EP4 and cytokeratin and stained negative for CD4, CD45 and vimentin.48 To establish a cell culture system of polarized human uterine, Fallopian tube and endocervical epithelial cells with both apical and basolateral compartments, primary cells were cultured in human extracellular matrix (Becton Dickinson, Franklin Lakes, NJ)-coated Falcon cell culture inserts in 24-well culture plates (Fisher Scientific, Pittsburgh, PA). For these experiments, apical and basolateral compartments
contained 300 and 850 μl of complete medium, respectively. In order to keep the culture selleck chemical conditions similar, the same procedure was followed for culturing the squamous ectocervical epithelial cells, which do not polarize. Akt inhibitor The medium was changed every 2 days. The cells were treated with 25 μg/ml of TLR3 agonist Poly(I:C) (Invivogen) for 24 hr, after which apical and basolateral conditioned media
(CM) were collected, centrifuged for 5 min at 10 000 g and stored at −80° until use. Tight junction formation of cultured epithelial cell monolayers was assessed by periodically measuring transepithelial resistance (TER) using an EVOM electrode and Voltohmmeter (World Precision Instruments, Sarasota, FL), as described previously.49 TER is a functional measurement of the integrity of tight junctions in an epithelial cell monolayer. The
presence of non-epithelial cells in the culture interferes with the formation of tight junctions and therefore prevents an increase in TER. TER is also an indicator for the purity of the epithelial monolayer. The concentrations of Trappin-2/Elafin in the to apical and basolateral supernatants from primary FRT epithelial cells and CVL from HIV-positive and HIV-negative women were determined using an enzyme-linked immunosorbent assay (ELISA) Duoset kit (R&D Systems, Minneapolis, MN), according to the manufacturer’s protocol. This assay measures both Trappin-2 and Elafin. The amounts of Trappin-2/Elafin were measured based on a standard curve after measuring the absorbance at 450 nm on an ELISA reader (Dynex, Chantilly, VA). Real-time reverse transcription–polymerase chain reaction (RT-PCR) was performed using a two-step protocol, as described previously.50 Total RNA was isolated from cells using TRIzol Reagent according to the manufacturer’s recommendations (Invitrogen Life Technologies) and purified by elution through RNeasy columns (Qiagen, Valencia, CA). Coincident with RNA purification was on-column DNAse digestion using the RNAse-Free DNAse set (Qiagen). For each specimen, 400 ng of total RNA was reverse-transcribed using the iScript complementary DNA (cDNA) synthesis kit (Bio-Rad, Hercules, CA), according to the manufacturer’s recommendations, in a 20 μl volume.