Supplementary Figure 4: Chimeric Cx3cr1^CreER–YFP:R26^Td monocytes retain the ability to distinguish resident macrophages from recruited macrophages post myocardial infarction in the setting of parabiosis and shielded irradiation.

a) Cx3cr1^CreER–YFP:R26^Td mice (Donor CD45.2) were fed tamoxifen (TAM)-containing chow at 3 weeks of age for 1 week, TAM was then discontinued for additional 10 weeks. At this time, Cx3cr1^CreER–YFP:R26^Td mice were paired with recipient CD45.1 mice. Two weeks after pairing, recipient CD45.1 mice were infarcted by surgical ligation of the left anterior descending artery (MI). At day 5 post-MI, blood and cardiac tissue (ischemic zone) was quantified for %Td⁺ in chimeric CD45.2 macrophages (MFs) (CD45⁺CD64⁺CD11b⁺) and blood monocytes (CD115⁺CD11b⁺) in the CD45.1 recipient mouse. N=3. One experiment. b) Bone marrow was isolated from adult Cx3cr1^CreER–YFP:R26^Td mice (Donor BM, CD45.2) treated as in panel (a). The bone marrow cells were transplanted into non-lethally irradiated CD45.1 mice (Recipient) that had a lead shield covering the heart and thoracic cavity to minimize loss of resident cardiac macrophages. Following 6 weeks of reconstitution, surgical MI was performed on the recipient CD45.1 mouse and it recovered for an additional 4 weeks. Blood and cardiac tissue (separated into ischemic and remote zone, see Fig. 4a) was taken for flow cytometric analysis. A subset of mice was given a pulse of TAM (i.p.) 2 days prior to isolation to confirm the responsiveness of the CD45.2 cells to activate the TAM-inducible Td reporter compared to No TAM controls. Gated on total CD45.2⁺ blood monocytes (CD115⁺CD11b⁺) and total CD45.2⁺ cardiac macrophages (CD64⁺CD11b⁺) in the CD45.1 recipient and %Td⁺ was quantified. N=3; one experiment. For all: center value, mean; error bars, SEM.