An Assay to Detect Autoantibodies in Human Serum using a Hippocampal Neuronal Culture
Transcript
Take rat embryo hippocampi.
Add trypsin. Incubate to mediate tissue matrix breakdown, loosening hippocampal cells.
Dilute with a buffer. Incubate for continued enzymatic digestion with trypsin.
Next, transfer the tissue mass to a media-containing tube. Pipette repeatedly for mechanical dissociation into single cells.
Seed cells on poly-L-lysine-coated coverslips within culture plates.
Incubate. Hippocampal cells adhere to the coated surfaces. The neurons develop a lamella and extend minor neurites.
Further, the neurites grow with axonal and dendritic processes, forming a neuronal network.
Mature neurons express synaptic excitatory receptors, N-Methyl -D-aspartate receptors, or NMDARs.
Post-incubation, wash the coverslips with media.
Add a diluted autoimmune encephalitis patient serum. The autoantibodies bind to neuronal NMDARs.
Wash with buffer and then fix the cells.
Add fluorophore-labeled secondary antibodies that bind to the NMDAR-bound autoantibodies.
Wash with buffer. Place the coverslip over mounting media containing DAPI to stain nuclei.
Microscopically observe fluorescence signals on neurons, indicating the presence of autoantibodies in the serum.
