Efficient technique for immortalization of murine microglial cells relevant for studies in murine models of multiple sclerosis Julie K. Olson a , Scott S. Zamvil b , Stephen D. Miller a, * a Department of Microbiology-Immunology and Interdepartmental Immunobiology Program, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611, USA b Department of Neurology, University of California San Francisco, San Francisco, CA, USA Received 25 February 2003; received in revised form 5 May 2003; accepted 7 May 2003 Abstract Microglia are macrophage-like cells that populate the central nervous system (CNS) and become activated upon injury or infection. Microglia have been implicated as playing critical roles in various CNS diseases including multiple sclerosis (MS), a human autoimmune demyelinating disease, as well as in other neurodegenerative diseases. Two well-characterized models of MS, relapsing experimental autoimmune encephalomyelitis (R-EAE) and Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease, are inducible in SJL mice and model the relapsing  /remitting and chronic-progressive forms of MS, respectively. These models are useful for the study of the mechanisms of initiation, progression, and therapy of the disease. Currently, a major limitation to studying the functions of microglia in these murine models of MS is the restricted number of cells capable of being isolated from the CNS of neonatal mice and propagated in culture. The current studies describe the preparation of SV-40 large T antigen-immortalized mouse microglia lines, M4T.4 and M4T.6, from the SJL/J mice. The immortalization technique was very efficient requiring only 6 weeks to develop long-term, highly replicating cell lines. The resulting microglia cell lines remain quiescent, but are induced to express various immune cytokines and to function as efficient antigen presenting cells upon activation with IFN-g or infection with TMEV. Thus, the SV-40 large T antigen immortalized microglia lines react to innate and infectious stimuli similar to primary microglia isolated from neonatal mice, but are more easily maintained in culture. This technique should allow for the efficient cultivation of large numbers of microglial cells from a variety of disease-relevant mouse strains, including knock-out and transgenic mice. # 2003 Elsevier B.V. All rights reserved. Keywords: Immortalization; Microglial cell line; Antigen presentation; Theiler’s virus 1. Introduction Microglia are the major antigen presenting cell population of the central nervous system (CNS) and share many characteristics with peripheral macro- phages. Microglia develop from mononuclear cells in the peripheral blood that originate in the bone marrow (Ling et al., 2001). These cells populate the CNS during embryonic development shortly after vascularization. The microglia exist in the developing CNS as an amoeboid form and serve as phagocytes clearing cellular debris during development (Ling et al., 2001). As development of the CNS is completed, microglia convert to a ramified form and revert to resting cells. Microglia compose 5  /20% of the glial population in the CNS and are more predominant in the gray matter than the white matter (Cuadros and Navascues, 2001). Microglia represent the first line of defense for the CNS and remain quiescent until injury or infection of the CNS rapidly activates them to become immune competent cells (Streit, 1995; Kreutzberg, 1996). They have been shown to function in both innate and adaptive immune responses to infectious challenge (Shrikant and Benve- niste, 1996; Olson et al., 2001). Microglia are present in areas of inflammation in the CNS and have been implicated in many human CNS diseases, including multiple sclerosis (MS), HIV, Alzhei- * Corresponding author. Tel.:  /1-312-503-7674; fax:  /1-312-503- 1339. E-mail address: s-d-miller@northwestern.edu (S.D. Miller). Journal of Neuroscience Methods 128 (2003) 33 /43 www.elsevier.com/locate/jneumeth 0165-0270/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0165-0270(03)00145-6