Figure 2. Isolation-dependent approaches for microglial proteomics.

Acute isolation of microglia and other brain cell types requires fresh brain tissue that undergoes mechanical or enzymatic dissociation to generate a heterogenous single-cell suspension that can be prepared for MACS, FACS, or immunopanning methods. For MACS, the single-cell suspension is incubated with a magnetic microbead conjugated to an antibody that binds a cell surface receptor. Then, a magnet is used to select for the desired cell type and the unbound cells are washed away. The magnetically bound cells are released and collected for downstream analysis. For FACS, the single-cell suspension is incubated with a fluorophore-conjugated antibody specific to a cell surface receptor. Following, the desired cell type is sorted based on size and fluorescent signal. The sorted cells are collected for downstream analysis. For immunopanning, single-cell suspensions are plated on a cell culture dish coated with an antibody specific to a cell surface receptor. The unbound cells are washed away, and the bound cells are collected for downstream analysis.