Fig 2: Changes in cellularity based on cell preparation method. Cx3cr1-NuTRAP brains were hemisected and single-cell suspensions were generated by three different methods. Cell preparations were compared via flow cytometry and transcriptomic analyses. The three cell preparation methods were also compared with whole tissue using transcriptomic analysis. Basic sequencing metrics and all source data for Figure 6 are provided in Extended Data Figure 6-1. Gene body coverage plots showed greater 3' bias in the Tissue- and Dounce-Input groups than the Cell-Input (+/-) Inhibitors groups (Extended Data Fig. 6-2). A, Schematic of experimental design presented in this figure. B, Flow cytometry on cell type-specific markers for microglia (CD11b+CD45+), astrocytes (ACSA2+), oligodendrocytes (O4+), and neurons (CD24+) for each of the cell preparation methods (one-way ANOVA, Tukey’s post hoc test, ***p < 0.001). Representative gating strategies for flow cytometry are provided in Extended Data Figure 6-3. C, Heatmap of cell type-specific markers for microglia, astrocytes, oligodendrocytes, neurons, and endothelial cells. D, CIBERSORTx cellularity estimates based on whole-transcriptome RNA-Seq from whole brain tissue and each of the cell preparation techniques. E, Comparison of the number of eGFP+ singlets in each half brain for each of the cell preparation methods (one-way ANOVA, Tukey’s post hoc test, ***p < 0.001). There was no overall difference in cell viability between different cell preparation methods. eGFP+ cells had slightly higher viability in the Cell-Input (+/-) Inhibitors than the Dounce-Input method (Extended Data Fig. 6-4). Boxplots represent median, Q1, Q3, minimum, and maximum of each dataset. Created with BioRender.com.

Fig 3: Comparison of purity and yield among microglial cell isolation techniques. A, Schematic of the experimental design. Cx3cr1-NuTRAP brains were enzymatically and mechanically dissociated to create a single-cell suspension. Different microglial sorting techniques were compared with Cell-Input for purity, yield, and transcriptomic signatures. B, Representative flow cytometry plots of immunostained single-cell suspensions from input and after each of the sorting strategies shows a distinct population of eGFP+ cells (identified as Cx3cr1+ microglia) and CD11b+CD45+ cells (identified as microglia per traditional cell surface markers, as shown by IHC in Extended Data Fig. 1-2). Assessment of the loss of eGFP in circulating CD11b+CD45+ cells 60 d after Tam induction (Extended Data Fig. 1-3) and the relative levels of CD45mid and CD45high cell populations from the CD11b+CD45+ each sort method and cell input from brain (Extended Data Fig. 1-4) were also performed to further characterize the sort fractions. Representative plots of the gating strategies used for the Tyto sort (Extended Data Fig. 1-5), FACSAria sort (Extended Data Fig. 1-6), and flow cytometry (Extended Data Fig. 1-7) are given as supplements. C, D, All sort positive fractions were enriched for (C) eGFP+ singlets and (D) CD11b+CD45+ singlets (as compared with input; two-way ANOVA, main effect of TRAP fraction, *p < 0.05, ***p < 0.001; Extended Data Fig. 1-1). When comparing positive fractions, the autoMACS positive fraction had lower % eGFP+ singlets as compared with all other sort methods. FACSAria had higher percentage of eGFP+ singlets than all other sort methods. FACSAria had higher percentage of CD11b+CD45+ singlets as compared with all other sort methods positive fractions (two-way ANOVA, Tukey’s post hoc test, *p < 0.05). E, Microglial yield was significantly higher in the MACSQuant Tyto positive fraction as compared with the autoMACS to MACSQuant Tyto and FACSAria positive fractions (one-way ANOVA, Tukey’s post hoc test, #p < 0.01). Created with BioRender.com.

Fig 4: Cellular binding and internalization assay using mouse NPC. (a) Cellular dot plot after staining with CD146 and CD45. X axis (Pacific Blue) and Y axis (FITC) shows CD45 and CD146, respectively. CD146 high/CD45 low, CD146 low/CD45 low, and CD146 low/CD45 high population were named as P1, P2, and P3, respectively. (b) Histogram of the FcγRIIB expression in each population. X axis (APC) shows the fluorescence of Alexa Fluor 647. Red shows staining with anti- FcγRIIB antibody and blue shows unstaining. (c) Concentration-dependent binding of the antibody in P2. The antibody was incubated at 4 °C for 60 min, and fluorescence was detected by FCM. The data were fitted with the receptor-ligand binding equation. (d) Concentration-dependent internalization rate of the antibody in P2. The antibody was incubated at 37 °C for 5 min. The data were fitted with the Michaelis–Menten equation. Each point shows the mean ± SD (n = 3).

Fig 5: Adipose remodelling was no different in human IGF‐1 receptor endothelial overexpressing mice (hIGFREO) in the setting of high‐fat diet (HFD)‐induced obesity A–C(A), Histological examination of brown and white adipose tissue in hIGFREO compared with WT after 8 weeks of HFD. Scale bar = 300 µm. (B), hIGFREO mice have increased abundance of smaller adipocytes than WT. (C), There is no difference in average size of adipocytes in brown adipose tissue of hIGFREO compared with WT using haematoxylin and eosin stain (n = 6 per genotype).DRepresentative confocal microscopy images of whole mount white and brown adipose tissue. Adipocytes stained with LipidTOX (green) and endothelial cells stained with Isolectin B4 (IB4‐647) (red). Scale bar = 50 µm (n = 4–5 mice per group).EThere is no difference in vascular density in white or brown adipose tissue from hIGFREO compared with WT after 8 weeks of HFD (7 per genotype).FMacrophage infiltration, shown by crown‐like structure (CLS) analysis, was similar in hIGFREO and WT after 8 weeks of HFD (n = 7–8 mice per group).G–IThere is also no difference in resident adipose CD45+ cells, CD11b+ cells or F4/80 in hIGFREO compared with WT after 8 weeks of HFD (n = 7–8 mice per group). Data information: Data shown as mean ± SEM and individual mice are shown as data points, P < 0.05 taken as statistically significant using Student’s t‐test and denoted as * and ** for P < 0.01. ns denotes not significant.