An Assay to Detect Autoantibodies in Human Serum using a Hippocampal Neuronal Culture

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.