An optimized method to process mouse CNS to simultaneously analyze neural cells and leukocytes by flow cytometry

• Digestion with collagenase D + DNAse I is optimal to isolate neural and immune cells from mouse CNS.
• One step Percoll is superior to Percoll gradient to isolate neural cells.
• Optimized technique increases cell yield while preserving cell integrity.

BackgroundFlow cytometry is an efficient and powerful technique to characterize and quantify numerous cells. However, the strengths of this technique have not been widely harnessed in neurosciences due to the critical step of CNS tissue preparation into a single cell suspension. Previous reports assessed either neural cells or infiltrating leukocytes but simultaneous detection has not been extensively implemented. We optimized CNS tissue preparation for flow cytometry analysis.New methodWe subjected CNS tissue from individual adult mice to different digestion protocols and Percoll™ methods. We quantified and characterized by flow cytometry neural cells (neurons, oligodendrocytes, microglia) and leukocytes (macrophages, T lymphocytes).ResultsThe one step Percoll™ method significantly increased cell yield compared to the gradient Percoll™ method. The collagenase D + DNase I digestion led to the maximal cell number recovery while preserving cell marker (O4, NeuN, CD45, CD11b, CD3, CD4, CD8) integrity compared to papain, trypsin digestion, and no digestion. The combination of collagenase D + DNase I digestion and one step Percoll™ method was optimal for the recovery and analysis of cells from the CNS of naïve and experimental autoimmune encephalomyelitis (multiple sclerosis model) mice.Comparison with existing method(s)Although flow cytometry does not reveal CNS localization, this technique allows concurrent quantification of multiple parameters. In contrast to other protocols, our novel method simultaneously analyzes neural and immune cells in individual mice in healthy and pathological conditions.ConclusionsWe strongly believe that the field of neurosciences will benefit from an optimal use of flow cytometry to elucidate physiological and pathological processes.