Fig. 4: Assessment of false-positive signal in microglia and border macrophages using “sniffer cells”.

a Experimental schematic for assessment and abrogation of false-positive signal using “sniffer cells” and inhibitors of engulfment and lysosomal degradation. “Sniffer cells” for false SYN1 signal were introduced by mixing brain tissue from SYN1-KO mice (crossed with Ubi-GFP mice for identification) with brain tissue from WT mice. The samples were incubated with or without the cocktail of inhibitors and enzymatically digested with collagenase IV at 37 °C. Samples were then processed for flow cytometric analysis of SYN1. b Overview of the pharmacological inhibitor cocktail targeting engulfment and lysosomal degradation indicating the respective cellular processes targeted by each inhibitor. c SYN1-KO “sniffer cells” (shown in green) were introduced to reveal false-positive signals due to ex vivo SYN1⁺ contamination. The difference between WT cells and “sniffer cells” in the mixed samples without inhibitors is that the SYN1 signal in WT cells can be due to a combination of SYN1⁺ material engulfed in vivo (true signal, shown in pink) and ex vivo SYN1⁺ contamination (false-positive signal, shown in blue). In contrast, any SYN1 signal in the “sniffer cells” can only be due to ex vivo SYN1⁺ contamination and represents a false-positive signal. Treatment with the inhibitor cocktail is designed to reduce/prevent false-positive signals due to ex vivo engulfment, while SYN1 signal from in vivo engulfment (true signal) should be unaffected. SYN1 signal was assessed in both microglia (d) and BAMs (e). The flow cytometry plots show the SYN1 signal for WT and “sniffer cells” (S) in the mixed samples (separated on the x-axis based on GFP expression). The gates for SYN1⁺ cells are based on the fluorescence of their respective isotype controls (<1% cells in positive gate). Cells were gated on live, single cells, CD45⁺, CD11b⁺, CD64⁺, and GR1^- and microglia were further gated on CX3CR1^high and P2Y12^high while BAMS were gated on CD206^high and CD38^high. n = 4 mice, all males, per condition. The mean fluorescent intensity (MFI) for SYN1 with isotype controls subtracted is depicted for all microglia (d) and BAMs (e) independent of the gating shown on the plots. Error bars indicate standard error of the mean. Statistical analysis: One-way ANOVA, Bonferroni’s multiple comparisons test. Source data provided.