Class-specific monocytes are selected based on physiological challenges. (A) Representative FACS plots showing changes in the proportions of monocytic classes after 3 hours of bacterial infection in vivo (n = 3 independent experiments, using 3 mice for each). (B) Experimental scheme of functional assays (Figure 7C-D; supplemental Figure 7B-C) that evaluate selection vs induction of primary murine monocytes upon bacterial infection. The fluorophores labeled for each class of monocytes were used in different combinations as well. (C-D) Bar graphs showing the percentage of primary monocyte subsets left in pooled samples after live E coli and S aureus infection, respectively, in vitro (n = 3 independent experiments, using 3 mice for each). (E-F) Bar graphs showing the number of ER-Hoxb8 monocytes found in the BM (black) or peritoneal cavity (dark gray and light gray) of Ccr2 KO mice after heat-killed E coli and S aureus infection, respectively, in vivo (n = 3 mice). The cells from the BM were used as baseline to define the proportion of each injected monocyte class. (G-H) Bar graphs showing percentage of primary monocyte subsets left in pooled samples and F4/80 expression levels after 24 hours of live bacterial infection into 5FU-treated mice in vivo, respectively (n = 3 independent experiments, using 3 mice each). Statistical tests were done against phosphate-buffered saline (G). (I) Bar graphs showing phagocytosis efficiency in secondary exposure to live bacteria. (J) Survival curves of sublethally irradiated mice with injection of individual ER-Hoxb8 clones followed by infection with LPS (n = 8 mice with 2 independent experiments). (K-L) Survival curves of lethally irradiated mice with injection of ER-Hoxb8 monocytic clones followed by live bacterial infection (n = 10 mice with 2 independent experiments). A mixed model analysis of variance was used for the statistical test (C-D,G-H). ∗P < .05; ∗∗P < .01; ∗∗∗P < .005.