Figure 2. Loss of Cx3Cr1 results in a significant increase of Ly-6C “inflammatory” monocyte infiltration into GBM.

A) Representative dot plots that are gated on CD11b⁺CD45⁺ cells, with red and green circles defining CD11b⁺CD45^hi (blood-derived macrophages) and CD11⁺CD45^lo/int (resident brain microglia). Total population of CD11b⁺CD45⁺ is considered as 100%, and they are further gated on Ly-6C and F40/80 positivity for tumors from the three genotypes (upper panel). The CD45⁺CD11b⁺ population is further gated for Ly-6C and Ly-6G positivity to distinguish monocytes from neutrophils and further gated for GFP (CX3CR1), which shows that while inflammatory monocytes are positive for GFP, neutrophils are negative. B) Dot plots represent the percentage of CD45^hi population in the total CD11b⁺CD45⁺ population of tumors from the three genotypes. Although there is a trend towards an increase in CD45^hi in tumors from Cx3cr1^GFP/GFP mice, it does not reach statistical significance (n=4, 8 and 5 individual tumors for B6, Cx3cr1^GFP+ and Cx3cr1^GFP/GFP, respectively). Tumor sizes were chosen close to the end-point of survival to ensure similar sizes in the three different genotypes. C) Dot plots represent the percentage of Ly-6C^hi monocytes in the total CD11b⁺CD45⁺ population in tumors from the three different genotypes (each dot corresponds to one animal). A one-way ANOVA with Tukey's multiple comparisons test was performed and demonstrated that there was a statistically significant increase in the percentage of Ly-6C^hi monocytes in tumors from Cx3cr1^GFP/GFP animals compared to B6 or Cx3cr1^GFP+ animals (*p < 0.05 and **p < 0.01, correspondingly). D) Dot plots represent the percentage of Ly-6G⁺ neutrophils in tumors from the three different genotypes showing that loss of one or both copies of Cx3cr1 had no impact on neutrophil infiltration into GBM.