Despite the fact that microglia cells were first described almost a century ago, microglia-derived immortalized cell lines have only been established in the last two decades. One should be aware of their limitations but also of their advantages. Cell lines offer a potentially powerful tool to investigate some functional aspects of microglia. Cell culturing of human and murine microglia cell lines will be described in this chapter. It includes a presentation of equipment needed, cell culture medium and supplements, cell culture monitoring, and a protocol describing the steps for subculturing of microglia cell lines.