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To define a clear ex vivo flow cytometric phenotype for adult human microglia that would distinguish it from all other macrophage lineage cells in the central nervous system (CNS) or blood, and to utilize this phenotype to examine the activation state and CD4 expression of microglia freshly derived from CNS tissue of HIV-positive patients and those with other neurological diseases.Fresh human CNS tissue from both HIV-uninfected and HIV-infected individuals was obtained by biopsy or resection, and cells isolated immediately, labelled for flow cytometry and analysed.A Percoll density gradient isolation technique and phenotypic characteristics used for rodent microglia were applied and modified.Resident microglia could clearly be defined by the flow cytometric phenotype CD45low CD4−CD11b+ CD11chigh major histocompatibility complex (MHC) class II+ CD26− CD14−. Assuming normally low-level MHC class II expression in the healthy CNS, it was likely that MHC class II positivity reflected underlying pathology necessitating biopsy or resection and appeared to be a ‘leaky’ activation marker. Microglia activation was observed in specimens from only six (35%) out of 17 HIV-uninfected but all four (100%) HIV-infected patients, defined strictly as any level of upregulation of CD4 expression, to produce the phenotype CD45low/medium CD4low CD11b+ CD11chigh MHC class II+/++ CD26− CD14−. Where examined by immunohistology, CD68 was also upregulated in these cases.When activated in situ, microglia express low levels of CD4 and this is always seen in tissue from HIV-infected patients. Using the flow cytometric phenotype established here, microglia from HIV-infected tissue can now be isolated in pure form and studied directly ex vivo.