Establishment and Characterization of Primary Adult Microglial Culture in Mice

Abstract

Microglial cells account for approximately 12-15 % of the cells in the central nervous system (CNS). Microglial cells are polarized by pathological stimuli such as cytokines, chemokines, and growth factors, and play important roles in the deterioration and repair of the CNS. Here, we established cultures of primary microglial cells isolated from the brains of adult C57/BL6 mice using Percoll density gradients. The cells were cultured and stained with antibodies against CD11b, glial fibrillary acidic protein, myelin basic protein and NeuN to determine microglial, astroglial, oligodendroglial, and neuronal cells respectively. Moreover, the cells were exposed to interferon-γ (IFNγ) plus interleukin-1β (IL-1β) or IL-4 for 24 h to demonstrate the activating phenotypes with inducible nitric oxide synthase (iNOS), Ym1, and Iba-1 immunoblotting. At least 95 % of the cultured cells were CD11b-positive and -negative for astroglial, neuronal, and oligodendrocyte markers. IFNγ plus IL-1β treatment resulted in classical activation, which was represented by an increase in iNOS. The cells also displayed alternative activation, which increased Ym1 when treated with IL-4. The present study indicates that the microglial cells isolated as described here are a useful tool for elucidating adult microglial function.

Dysfunction of cerebellar microglia in Ataxia‐telangiectasia

“…Between 5% and 15% of the cells in the central nervous system (CNS) are microglia (Herculano‐Houzel, 2014; Ohtaki et al, 2013). These cells are derived from early embryonic myeloid progenitors of the hematopoietic system.…”

RNA‐sequencing reveals oligodendrocyte and neuronal transcripts in microglia relevant to central nervous system disease

“…The development of therapeutic strategies that target microglia‐produced factors requires the identification of microglia transcripts in both health and in the context of neurological disease. Several methods are now typically employed for these studies, including microglia culture in vitro (Hassan et al, ; Ohtaki et al, ; Szabo and Gulya, ), fluorescence‐activated cell sorting (FACS; Hassan et al, ), laser capture microdissection (LCM; Waller et al, ), and ribosome messenger RNA (mRNA)‐trap (BacTRAP and Ribo‐Tag) technologies (Heiman et al, ; Sanz et al, ). While each of these approaches has its strengths and limitations, there are two major barriers to these discovery efforts: First, the RNA routinely isolated from microglia is in low abundance and frequently of low quality, necessitating new methods for RNA isolation and analysis (Pong et al, ; Tariq et al, ).…”