This article describes a method for isolating and culturing rat embryo hippocampal neurons. The process includes enzymatic and mechanical dissociation of tissue, followed by immunostaining to detect NMDAR autoantibodies.
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.
For the enzymatic dissociation of the hippocampus at 1 milliliter of 2.5% trypsin in the 50-milliliter tube containing the hippocampi, after bringing the volume in the tube to 5 milliliters with HBSS, incubate the tube for 15 minutes in the water bath at 37 degrees Celsius. To further dilute the trypsin, add 10 milliliters of pre-heated HBSS, and place the tube back in the water bath set at 37 degrees Celsius for 5 minutes.
Next, use a 1,000-microliter micropipette to transfer the hippocampi appearing as a mucous mass to a 50-milliliter tube, and incubate the tissue with 6 milliliters of pre-heated HBSS for 5 minutes, as demonstrated. To dissociate the tissue mechanically, transfer the hippocampal mass to a 2-milliliter tube with conical bottom. After adding 1 milliliter of pre-heated DMEM media, homogenize the pellet with a 1,000-microliter micropipette by gently aspirating up and down.
Ensuring not to create bubbles, repeat the up-and-down aspiration 10 to 20 times with a pre-pulled glass pipette, with its tip in contact with the conical bottom of the tube, until the mixture is translucent. Count the cells before distributing them evenly on a 3.5-centimeter dish with crossed-shaking movements. Once done, place the dish in the incubator with 5% carbon dioxide.
For fluorescent immunostaining of 14 days in-vitro cells, add the sample containing anti-NMDAR antibodies to the coverslips, and incubate at 37 degrees Celsius and 5% carbon dioxide. After one hour, treat the sample with 4% formaldehyde in PBS, as a fixation solution, at room temperature for 5 minutes. Then wash the sample thrice with PBS for 5 minutes each, followed by addition of secondary antibody goat anti-human Alexa Fluor 488 at 1 to 1,000 dilution for 1 hour at room temperature. Next, mount the coverslips containing the sample with a liquid-mounting medium, and aspirate any remaining liquid.
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