Related Experiment Video
Updated: Aug 9, 2026
Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients
Published on: February 3, 2011
Isolation of brain parenchymal lymphocytes for flow cytometric analysis. Application to acute viral encephalitis
1Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Insights
A new method isolates mononuclear cells from inflamed mouse brains for flow cytometry analysis. This technique enables detailed study of central nervous system inflammation, offering more insights than traditional methods.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Central nervous system (CNS) inflammation involves mononuclear cell infiltration into brain parenchyma and cerebrospinal fluid (CSF).
- Sindbis virus (SV) encephalitis in mice serves as a model for studying CNS inflammation.
- Existing methods like immunohistochemistry provide limited quantitative analysis of inflammatory cells.
Purpose of the Study:
- To develop a novel strategy for isolating mononuclear cells from inflamed mouse brain parenchyma.
- To enable quantitative flow cytometric (FCM) analysis of these isolated CNS inflammatory cells.
- To provide a more sophisticated analytical approach compared to traditional tissue section staining.
Main Methods:
- Collection and homogenization of perfused brain tissue from SV-infected mice.
- Mild enzymatic digestion followed by sedimentation at unit gravity to remove debris.
- Centrifugation over a modified density gradient to separate intact cells from tissue fragments.
Main Results:
- Isolated cells are suitable for direct staining with monoclonal antibodies and FCM analysis.
- The method yields representative samples of the cellular infiltrate in the CNS.
- Data generated correlates well with previous immunohistochemical findings.
Conclusions:
- The developed strategy effectively isolates mononuclear cells from inflamed mouse brains for FCM analysis.
- This technique offers a more sophisticated and quantitative approach to studying CNS inflammatory cells.
- The method enhances the understanding of cellular responses during neuroinflammation.
Abstract:
A strategy for the isolation of mononuclear cells from the brain parenchyma of mice with ongoing central nervous system (CNS) inflammation has been developed in order to permit flow cytometric (FCM) analysis of these cell populations. Sindbis virus (SV) encephalitis in mice is characterized morphologically by an infiltration of mononuclear cells into both brain parenchyma and cerebrospinal fluid (CSF). Perfused brain tissue from infected animals is collected, homogenized, and subjected to a mild enzymatic digestion. A sedimentation at unit gravity is performed to remove any large particulate debris, and the remaining tissue is then centrifuged over a modified density gradient which separates intact cells from smaller tissue fragments. Cells collected directly from these gradients can be stained with monoclonal antibodies and analyzed by FCM without further manipulation. Data generated by this method correlates with previous studies of SV encephalitis using immunohistochemical analysis of brain tissue sections to quantify mononuclear cell types. This suggests that representative samples of the cellular infiltrate are obtained using this technique. The approach however, offers the possibility of more sophisticated and quantitative analyses of CNS inflammatory cells which is unobtainable by tissue section staining.
