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

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 …

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