Extended Data Fig. 6: Analysis of synaptic engulfment by OPCs and microglia using flow cytometry.

(a) Representative gating strategy for OPCs and microglia. The cells were initially distinguished from debris by the expression of CD45 and PDGFRA proteins. Next, the CD45low and PDGFRAhi population were separately gated as follows: (Ai) single cells1 →single cells2 →viable cells →OPCs (PDGFRAhi+A2B5hi) and (Aii) microglia (Cd11bhi+CD45low). (Aiii) Back gating strategy was used as a reference for OPCs and microglia population between the different samples. n = 3 mice at P55. (b),(c) Flow cytometry plots showing immunoreactivity for SYNAPSIN and SNAP25 in both OPCs and microglia. An independent fluorescence minus one (FMO) control (grey) was used as a baseline to set the gate for the negative (SYN1/SNAP25 – negative) and positive (SYN1/SNAP25 – moderate and SYN1/SNAP25 – high) events. The highest mean fluorescence intensity in the microglia population was used to set the gate for the ‘high engulfers’ in OPCs (SYN1/SNAP25 – high, highlighted in magenta on the merged plot). (d),(e) Relative quantification of all positive events for SYN1 and SNAP25 in OPC and microglia populations. Two-tailed paired t-test, P > 0.05. Box plots show s.e.m., 25% quartile and median values. (f) UMAP showing a distinct OPC population with high immunoreactivity for SYN1 compared to the negative and moderate populations. All data represented is pooled data from three independent biological replicates.