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

Published: March 29, 2024

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) 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 st…

Representative Results

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 into 1. Hippocampal isolation, 2. Cell dissociation, and 3. Cell seeding. (B) Selection of recommended tools for hippocampal …

Declarações

The authors have nothing to disclose.

Materials

10 cm Cell culture dish Nunc 12-565-020
12 mm round coverslips Fisher NC9708845
20x NA 0.75 S Fluor air objective Nikon CFI Super Fluor 20X
3.5 cm Cell culture dish Nunc 12-565-90
6 cm Cell culture dish Nunc 12-565-94
B27 supplement Gibco 17504-044
Beaker 100 mL Pirex
Borax Sigma-Aldrich B9876
Boric Acid Sigma-Aldrich B0252
Curved forceps FST 11009-13
D-Glucose Sigma-Aldrich D9434
DMEM High Glucose (4.5 g/L), without L-Glutamine, without Phenol Red Capricorn DMEM-HXRXA
Female Wistar rat (18-days pregnant) Janvier
Fetal Bovine Serum (FBS) Biowest S181B-500
Fine-angled forceps FST 11251-35
Fine-curved forceps FST 11272-30
Fine-straight forceps FST 11251-23
FITC filter cube Nikon Standard Series
Forceps FST 11000-12
Goat anti-Human AF488 Invitrogen A11013
HBSS Capricorn HBSS-1A
HEPES Sigma-Aldrich H3375
Hibernate-E medium Gibco A12476-01
Horse Serum (HS) Thermofisher 26050088
Human anti-NMDAR antibody (CSF) Patient Sample
Human anti-NMDAR antibody (Serum) Patient Sample
ImageJ/Fiji NIH v1.50i
Inverted fluorescence microscope Nikon Eclipse TE2000-U
Phosphate-Buffered Saline Gibco 10010023
Poly-L-Lysine (PLL) Peptide international OKK-35056
Polystyrene ice tray
Precision spring-scissors FST 15000-08
ProLong Gold with DAPI (antifading mounting media) Molecular Probes P36941
Scissors FST 14068-12
Sodium pyruvate Biowest L0642-100
Stereo microscope Zeiss Stemi 2000
Surgery scissors FST 14081-09
Trypsin 2.5% Gibco 15090046
Water, sterile endotoxine free Sigma-Aldrich W3500

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An Assay to Detect Autoantibodies in Human Serum using a Hippocampal Neuronal Culture. J. Vis. Exp. (Pending Publication), e22096, doi: (2024).

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