Gating strategy for leukocyte subpopulations and TLR expression.

Leukocytes were selected in the forward-sidescatter plot (fig. 1A) and copied to a sidescatter-CD14 plot. Monocytes (CD14 positive cells), granulocytes (CD14 negative cells with high SSC) and lymphocytes (CD14 negative cells with low SSC) were gated (fig. 1B). CD14 positive cells were copied to a TLR2/TLR4 plot. Using the isotype control sample, gates were set in the TLR2/TLR4 plot so that at least 99% of the isotype controls were negative for TLR2/TLR4 expression (fig. 1C). This gate was then used to identify the percentages of TLR4/TLR2 double positive, TLR2 single and TLR4 single positive monocytes as well as their mean fluorescence intensity (MFI), in the antibody incubated samples (fig. 1D).