Data were expressed as ng of DNA of the targeted genus or species per μg of total DNA extracted from the vaginal sample. Bioplex immunoassay Cytokine levels were
determined using a multiplexed bead immunoassay. Prior to assay, vaginal samples were concentrated 10 times with Microcon spin devices (YM3, Millipore Corporation, Billerica, MA) and subsequently resuspended in Bio-Plex Assay Buffer. The levels of 27 immune-mediators, 15 cytokines (IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, selleck chemical IL-12(p70), IL-13, IL-15, IL-17, IFN-γ, TNFα), 7 chemokines (MCP-1, MIP-1α, MIP-1β, RANTES, Eotaxin, IL-8, IP-10) and 5 growth factors (PDGF-BB, FGF basic, G-CSF, GM-CSF, VEGF), were measured using the human ultrasensitive cytokine 27-plex antibody bead kit (Bio-Rad). Assays were performed in 96-well filter plates, as previously described . Briefly, the filter plate was prewetted with washing buffer (Bio-Rad) and the solution was aspirated from the wells using a vacuum manifold (Millipore Corporation). Microsphere beads coated with monoclonal antibodies against the different target analytes were added to the wells. Samples and standards were pipetted into the wells and incubated for 30 min with the beads. Wells were washed using a vacuum manifold (Millipore Corporation) and biotinylated secondary antibodies were added. After incubation for 30 min, beads were washed then incubated
for 10 min with streptavidin-PE conjugated to the fluorescent protein, R-phycoerythrin (streptavidin/R-phycoerythrin). After washing to remove the unbound streptavidin/R-phycoerythrin, the beads buy Epacadostat (a minimum of 100 per analyte) were analyzed in the Luminex 200 instrument (MiraiBio, Alameda, CA). The Luminex 200 monitors the spectral properties of the beads to distinguish the different analytes, while simultaneously measuring the amount of fluorescence associated with R-phycoerythrin, reported as median fluorescence intensity. The concentration of the samples was estimated from the standard curve using a fifth-order polynomial equation and
expressed as pg/ml after adjusting for the dilution factor (Bio-Plex Manager software version 5.0). Samples below the detection limit of the assay were recorded as zero, while about samples above the upper limit of quantification of the standard curves were assigned the highest value of the curve. The intra-assay CV including ultrafiltration and immunoassay averaged 19%. Concentrations of cytokines, chemokines and growth factors were then converted in pg of the target molecule per μg of total proteins present in the vaginal sample. Statistical analysis Statistical analysis was performed using SigmaStat (Systat Software, Point Richmond, CA). For each subject, variations of the DGGE profiles related to the time points W33 and W37 were analyzed by Pearson correlation.