An Ex Vivo Technique to Induce Neuronal Death via Oligodendrocyte-Specific CD8+ T Cells
Published: August 31, 2023
Abstract
Source: Göbel, K., et al. An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices. J. Vis. Exp. (2015).
This video demonstrates an ex vivo technique to induce brain cell death mediated through cytotoxic CD8+ T cells. Upon obtaining brain slices, activated oligodendrocyte-directed CD8+ T cells are transferred onto the slices to target the myelin sheath and oligodendrocytes. Post-incubation, the damage to myelin and neuronal death can be assessed histologically in the regions of interest.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. General Comments for Mouse Experiments Keep the mice under pathogen-free conditions and enable them access to food and water ad libitum. NOTE: It is important to use age- and sex-matched mice in experimental groups because immunological patterns can vary with age and gender. <…
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The authors have nothing to disclose.
Materials
| 12-Well-plate | Corning | 3513 | |
| 2-Mercaptoethanol | Gibco | 31350-010 | |
| 2-Methylbutan | Roth | 3927.1 | |
| 70 µm strainer | Falcon | 352350 | |
| CaCl2 | Merck | 1.02382.0500 | Calcium chloride |
| CD8+-isolation kit | Miltenyi Biotech | 130-090-859 | |
| D(+)-glucose | Merck | 1.08337.1000 | |
| DMEM | Gibco | 31966-021 | Warm in 37 °C water bath before use |
| EDTA | Sigma | E5134 | |
| FCS | PAA Laboratories | A15-151 | Fetal calve serum |
| Gentamicin | Gibco | 15750-060 | |
| HEPES 1M | Gibco | 15630-050 | |
| IL-2 | Peprotech | 212-12 | |
| Isofluran | Abbott | 05260-05 | |
| KCl | Merck | 1.04933.0500 | Potassium chloride |
| KHCO3 | Sigma | P9144 | Potassium hydrogen carbonate |
| L-Glutamine | Gibco | 35050-038 | |
| MgSO4 | Merck | 1.05886.0500 | Magnesium sulfate |
| NaCl | Sigma | 31434 | Sodium chloride |
| NaH2PO4 * H2O | Merck | 1.06346.0500 | Sodium hydrogen phosphate |
| NaHCO3 | Merck | 1.06329.0500 | Sodium hydrogen carbonate |
| NaOH | Merck | 1.09137.1000 | Sodium hydroxide |
| NH4Cl | Sigma | 213330 | Ammonium chloride |
| Non-Essential Amino Acid | Gibco | 11140-050 | |
| OVA (257-264) | Genscript | RP10611 | Ovalbumin |
| PIPES | Sigma | P6757 | |
| Sukrose | Merck | 1.07687.1000 | |
| Tissue-Tek OCT | Sakura | 4583 |
