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

Abstract

Background: 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 reflect in vivo characteristics and functionality compared to immortalized cells; however, they are challenging to acquire and maintain.

New method: 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.

Results: 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 CD11b^hi and CD45^lo 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 production after treatment of lipopolysaccharide (LPS).

Comparison with existing methods: Our method allows for rapid isolation of both microglia and astrocytes in one protocol with relatively few resources, preserves cellular phenotype, and yields high cell numbers without magnetic or antibody sorting.

Conclusion: Here we show a novel, single protocol to isolate microglia and astrocytes from brain and spinal cord tissue, allowing for culturing and other downstream applications from the cells of animals of various ages, which will be useful for researchers investigating these two major glial cell types from the brain or spinal cord of the same rodent.

Keywords: Astrocytes; Culture; ELISA; Flow cytometry; IL-6; Immunocytochemistry; Isolation; Microglia.

Figure 1:

An illustration showing the experimental procedure for extracting microglia and astrocytes from rodent brain and spinal cord. Whole brain and spinal cord were collected from rodents and homogenized by passing through a 70 μm nylon mesh. Cell homogenates were subjected to centrifugation in percoll gradients and cell populations were collected from 70%-50% and 50%-35% interfaces. These extracted cell populations were then characterized by immunocytochemistry, flow cytometry, or ELISA. (Illustration created with Biorender.com).

Figure 2.

Immunocytochemistry for Iba1 (microglial marker) on cells isolated from 70%-50% SIP interface. A) Immunofluorescent labeling of Iba1, GFP, and DAPI in cells isolated from mice. (B) Immunofluorescent labeling for Iba1 and DAPI in cells isolated from rats.

Figure 3.

Immunocytochemistry for GFAP (astrocyte marker) on cells isolated from 50%-35% SIP interface. A) Immunofluorescent labeling of GFAP and DAPI in cells isolated from rats.

Figure 4.

Flow cytometry analysis on cells isolated from 70%-50% SIP interface. Isolated cells from mice (A) and rats (B) shows expression of Cd11b⁺ and Cd45^low (microglial marker) compared to respective unstained control.

Figure 5.

Flow cytometry analysis on the cells isolated from 50%-35% SIP interface. Isolated cells from mice (A) and rats (B) shows the expression of GLAST compared to respective unstained control.

Figure 6.

Isolated primary microglia and astrocytes cells stimulated with LPS, to assess functionality. (A) Microglia produce significant increase in level of IL-6 after LPS stimulation (1μg/ml, n = 4 animals per group, unpaired t-test ***p <0.0001. (B) Astrocytes produce significant increase in level of IL-6 after LPS stimulation (0.5μg/ml, n = 4 animals per group, unpaired t-test *p <0.05).