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