A dual reporter system reveals maturation of monocytes inside and outside of the lung vasculature.

<p>(<b>A</b>) Illustration of the <i>Cx3cr1</i>-eGFP-Cd11c-mCherry transgenic mouse model used for lineage tracing of monocyte to DCs. (<b>B</b>) Flow cytometry with absolute numbers (insets) and (<b>C</b>) normalized total counts of GFP⁺ and/or Cherry⁺ cells in saline, Ova or HDM-treated lungs within a CD45⁺CD11b⁺Siglec-F^-Ly6G^- gate. Data in C was normalized to input fluorescent polystyrene beads during lung digests. Data in B-C represent 3 independent experiments. (<b>D</b>) Experimental layout and results of intravenous labeling of blood monocytes as a pulse with intravenous beads (“pulse”), followed by tracking of bead+ lung-homing monocytes or monocyte-derived cells over 48 hours during the “chase” period. Flow cytometry data is plotted as a ratio of the GFP and mCherry channel with gates for ‘low’ set based on Saline at -24 hours, ‘med’ based on OVA at -24 hours and ‘hi’ representing populations that appear predominantly after 48 hours. Data represent 3 independent experiments. (<b>E</b>) Experimental layout and resultant flow cytometric analysis of intravenous labeling of cells with high molecular weight anti-CD45 antibodies 5 minutes prior to euthanasia. CD45⁺CD11b⁺Siglec-F^-Ly6G^- lung monocytic cells are plotted on the identical Cherry:GFP scale as (D), with the CD45 i.v. specifically labeling intravenous immune populations as delineated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165064#pone.0165064.s002" target="_blank">S2B Fig</a>. (<b>F</b>) Illustration of the extent of monocyte→myeloid maturation and concomitttant tissue distribution that is differentiated by ratiometric dual reporting of <i>Cx3cr1</i>-eGFPx<i>Cd11c-</i>mCherry mice. Data in D-F representative of at least three mice per condition.