Detection of tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages and microglia (TAMs) in ex vivo tumor specimens. (A) Flow cytometryic analysis and gating strategy used to detect TILs (CD3+CD45+ cells) and TAMs (CD11b+CD45+ cells) in brain. The initial gate excludes data points with extreme side and forward scatter properties. (B) The frequency of TILs and TAMs as obtained by flow cytometry was increased in ex vivo tumor specimen. Note that the TAMs shown here were detected by CD45. (C) Immunofluorescent staining of ex vivo tumor specimen revealed a large number of TAMs in the tumor specimen as compared to neighboring white matter. Rhodamine (red) staining for CD11b indicating TAMs, Hoescht (blue) staining for nuclei. The increased cellularity on the left side of the image is grossly visible tumor tissue, and the right side of the image is white matter without gross tumor involvement.