Apr 22, 2020

Isolation, culture, and downstream characterization of primary microglia and astrocytes from adult rodent brain and spinal cord

Journal of Neuroscience Methods
Nilesh M AgalaveMichael D Burton

Abstract

Neuroimmunologists aspire to understand the interactions between neurons, microglia, and astrocytes in the CNS. To study these cells, researchers work with either immortalized cell lines or primary cells acquired from animal tissue. Primary cells reflectin vivo characteristics and functionality compared to immortalized cells; however, they are challenging to acquire and maintain. Established protocols to harvest primary glia use neonatal rodents, here we provide a method for simultaneously isolating microglia and astrocytes from brain and/or spinal cord from adult rodents. We utilized a discontinuous percoll density gradient enabling easy discrimination of these cell populations without enzymatic digestion or complex sorting techniques. We found cells isolated from the percoll interface between 70%-50% were microglia, as they express ionizing calcium-binding adaptor molecule 1 (Iba1) in immunocytochemistry and CD11bhi and CD45lo using flow cytometry. Isolated cells from the 50%-30% interface were astrocytes as they express glial fibrillary acidic protein (GFAP) in immunocytochemistry and Glutamate aspartate transporter (GLAST)-1 using flow cytometry. Cultured microglia and astrocytes showed a functional increase in IL-6 product...Continue Reading

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Mentioned in this Paper

Immunocytochemistry
Flow Cytometry
Study
Central Nervous System
Brain
Calcium-binding protein-2
Isolate - Microorganism
In Vivo
IL6
Calcium-Binding Proteins

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