Isolation of primary microglia, T cells and B cells
Our core expertise is in the isolation of primary CNS-resident immune cells including microglia, T cells and B cells. Pure CNS-resident immune cells are isolated using a rapid procedure based on Percoll density gradient centrifugation followed by magnetic beads-positive selection or fluorescence activated cell sorting. Due to a short postmortem delay and rapid procedure the primary cells show preservation of gene expression and phenotype that reflects neurological diagnosis.
Our isolated primary cells can be provided fresh in culture medium for use in cellular assays, as well as frozen, or lysed, enabling downstream molecular characterization as demonstrated in multiple publications making use of NBB tissue (see Publication list below).
Microglia isolation using percoll density gradient centrifugation and magnetic beads-positive selection
Isolation and sorting of nuclei
Nuclei are isolated from frozen tissue from the NBB collection by staining with cell type specific markers such as IRF8+ (microglia) or NeuN (neurons) followed by fluorescence-activated nuclei sorting. This enables to perform cell-type specific RNA sequencing analyses and single-nucleus sequencing. By linking these molecular data to the neuropathological data from the NBB donors we support identification of alterations in RNA expression that link to a disease state. As such we strive to contribute to identification and validation of disease pathways and novel targets.
Procedure for fluorescence-activated nuclei sorting