Figure 7—figure supplement 2.

Macrophage populations were sorted by Fluorescence-Activated Cell Sorting (FACS) before transcriptomic analysis.

(A–L) Tg(mfap4:mCherry-F/tnfa:GFP-F) larvae were either infected with Salmonella (Sal-INF) or non-infected (Non-INF). (A–D) Gating strategy to isolate mfap4⁺-tnfa⁺ cell (M1-activated) populations at 4 hpi from whole Tg(mfap4:mCherry-F/tnfa:GFP) Sal-infected larvae using light-scattering characteristics. Representative dot plots showing gating strategy for first isolating live cells based on Sytox-Red staining (A, B) and then isolate singlet cells (C, D). Cells from WT (Unlabeled) (E), Tg(tnfa:GFP) GFP⁺ cells (F), and Tg(mfap4:mCherry-F) (mCherry⁺) (G) were used for gating. To increase the purity of the sorted populations, the gates have been designed so that they do not overlap. (H–K) Representative dot plots showing gating strategy to isolate mfap4⁺-tnfa⁻ (Inactivated or Non-M1-activated) macrophage populations at 4 hpi (H) and at 4 dpi (J–K) and to isolate mfap4⁺-tnfa⁺ (M1-activated) macrophage populations at 4 hpi (I). Gating strategy tables with numbers of cells per gate (#Events), the percentage of cells in the parental gate (%Parent) and in relation to the total percentage of cells (%Total). (L) FACS sorting was used to isolate mfap4⁺-tnfa⁻ cells (inactivated or non-M1 activated) and mfap4⁺-tnfa⁺ cells (M1 activated) at 4 hpi and 4 dpi. Percentage of macrophage populations sorted by FACS. Data of four replicates per condition pooled (except for the Non-INF condition at 4 dpi where only three replicates were used (mean % of cells± SEM)). Forward SCatter-Area (FSC-A); Side SCatter-Area (SSC-A); Forward SCatter-Width (FSC-W).