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CD4+ lymphocytes analysis was done using three-color flow cytometry with antibodies against human-CD95, a prototype cell death receptor, and the chemokine receptor-7 (CCR7) after gating for lymphocytes based on the forward and side scatter.
after gating for viable, single cells.
Percentages of apoptotic cells were determined after gating for EpCAM +/Annexin V + cells.
After treatment, cells were incubated with TLR2, TLR4, or isotype-matched IgG (eBioscience) depending on the cell treatment; 10,000 events were analyzed with the Becton-Dickinson Fluorescence-Activated Cell Sorter Bioanalyzer as described previously (11) after gating for CD14.
Monocytes from control and MetS subjects were incubated with anti-human TLR2 and TLR4 antibodies (InvivoGen) or isotype controls, and surface expression of TLR2 and TLR4 were analyzed using BD FACSArray (Franklin Lakes, NJ) after gating for CD14 as previously described (15, 16).
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The mean fluorescence intensity (MFI) of the PE channel was determined for each sample after gating on bacterial cells in the logarithmic FSC vs. SSC dot plot.
After gating, a negative population was defined using the histogram obtained for medium-only negative control samples.
After gating, a negative population was defined using the histogram obtained for media-only negative control samples.
After gating granulocytes, the percentage of CD45/41-positive CD45/41-positiveunted.
Analyses were performed after gating on anti-CD45 stained lymphocytes.
Lin− cells were gated for CLP (Flk2+IL7Rα+).
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