Brain and spinal cord tissues from mice perfused with PBS were digested with collagenase IV (Sigma-Aldrich) for 30 min at 37 °C, resuspended in 37 % Percoll, and loaded between a 30 and 70 % Percoll gradient. After centrifugation at 2000g for 20 min, CNS mononuclear cells were retrieved from the 37/70 % Percoll interface as previously done [38 (link)]. PMN- and MO-MDSC subsets were purified from CNS mononuclear cells by immunomagnetic separation using biotinylated anti-Ly6G and anti-Gr1 antibody and streptavidin-conjugated MicroBeads as mentioned above (Miltenyi Biotec). Cell purity was >95 % by flow cytometric analysis using anti-CD11b and Gr-1 antibodies.
Protocol detail
CNS Mononuclear Cell Isolation
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Anti antibody
Antibodies
Brain
Cd11b
Cell
Centrifugation
Collagenase
Flow cytometric
Immunomagnetic separation
Mdsc
Mice
Microbeads
Percoll
Spinal cord
Streptavidin
Tissues
Corresponding Organization :
Other organizations : Washington University in St. Louis, Hope Center for Neurological Disorders
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Variable analysis
independent variables
- Digestion of brain and spinal cord tissues with collagenase IV (Sigma-Aldrich) for 30 min at 37 °C
- Purification of PMN- and MO-MDSC subsets from CNS mononuclear cells by immunomagnetic separation
- Cell purity >95% by flow cytometric analysis using anti-CD11b and Gr-1 antibodies
- Perfusion of mice with PBS
- Resuspension of digested tissues in 37% Percoll
- Loading between a 30 and 70% Percoll gradient
- Centrifugation at 2000g for 20 min
- Retrieval of CNS mononuclear cells from the 37/70% Percoll interface
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