Single Cell Cytometry of Mouse Brain Tissue

Suspension mass cytometry, capable of measuring 50 or more markers per cell, proved advantageous for comprehensive phenotyping of single cells in tissue with high inherent fluorescence, such as the brain. The intricate nature of the brain, densely interconnected with extracellular material, and its diverse cell types presented challenges in reducing it to a single-cell suspension.

This study examined methods for producing single-cell preparations from enriched white and gray matter regions of adult mouse brain, evaluating mechanical dissociation, Accutase, and three gentle enzyme combinations with DNase, alongside Percoll gradient centrifugation to separate viable cells from debris. Live cell counts via hemocytometer and Trypan blue exclusion evaluated yield across enzyme conditions.

Mass cytometry assessed cellular composition post-representative dissociation, debris removal, and cryopreservation. Main proteins examined were beta III-tubulin for neurons, GFAP for astrocytes, OLIG2 for oligodendrocytes, CD31 for endothelial cells, CD24 for ependymal cells, P2Y12 for microglia, and CD45 for leukocytes. Mechanical dissociation combined with enzymatic methods (papain, Collagenase/Disapse, or Collagenase II plus DNase) yielded about 400,000 live cells per 100 grams of tissue.

The yield remained consistent across conditions and was similar between white and gray matter. Adding Percoll gradient centrifugation decreased viable cell recovery by an average of 88%, resulting in insufficient numbers for robust analysis of less common cell subsets. Mass cytometry identified six major non-neuronal cell populations: oligodendrocytes, astrocytes, endothelial cells, ependymal cells, microglia, and leukocytes.

The relative abundance of these populations remained generally unchanged by cryopreservation in both gray and white matter, although oligodendrocyte loss occurred in both tissues. Proliferation, assessed by Ki67 positivity, was absent across all cell populations, with low levels of phosphorylated STAT1 (p-STAT1) and p-STAT3 proteins.

Cryopreservation resulted in a modest increase in p-S6(S235/236) protein only in microglia. These findings establish standardized procedures for generating viable single-cell suspensions for cytometry that preserve the cellular diversity of the adult murine brain with minimal intracellular signaling disruption.