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

In This Article

Abstract

Source: Cunquero, M., et al. Hippocampal Neuronal Cultures to Detect and Study New Pathogenic Antibodies Involved in Autoimmune Encephalitis. J. Vis. Exp. (2022)

This video demonstrates a technique for identifying antibodies against specific antigens in a sample using a rat hippocampal neuronal culture. By exposing cultured neurons to patient samples containing autoantibodies targeting N-Methyl-D-aspartate receptors (NMDARs), the presence of autoantibodies bound to the neuronal surface is revealed through immunostaining.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

PART 1: Establishment of dissociated, fetal, rodent hippocampal neuronal cultures

1. Preparative steps

Poly-L-Lysine (PLL) coating of the plating surfaces (3 days before dissection)
1. Prepare the borate buffer by adding 2.38 g of boric acid and 1.27 g of borax to 500 mL of distilled water and stirring for 15 min until dissolved. Under a hood, sterilize the buffer solution by filtering (0.2 µm pore size), label, and store at room temperature ....

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Results

Hippocampal cell isolation and seeding diagram; shows enzymatic, mechanical dissociation steps.
Figure 1: Visual protocol for primary cultures of hippocampal neurons. (A) Flowchart showing the three parts of the protocol for preparing dissociated-cell cultures of hippocampal neurons from embryonic rats at E18. The protocol is divided i.......