SEC-based Isolation of Microglia-derived Extracellular Vesicles: A Technique to Isolate Extracellular Vesicles from Microglia Conditioned Culture Media
Published: April 30, 2023
Abstract
Source: Lemaire, Q. et al. Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment. J. Vis. Exp. (2020)
This video describes a method based on size-exclusion chromatography (SEC) to isolate extracellular vesicles or EVs derived from the brain immune cells―the microglia. The EVs act as molecular cargo to deliver mediators, which regulate an immune response. Therefore, the isolated EVs can be used as an in vitro model to study the microglia-neuron crosstalk during a neuronal immune response.
Protocol
1. Primary Culture of Microglia/Macrophages Primary culture of microglia Culture commercial rat primary microglia (2 x 106 cells) (see the Table of Materials) in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% exosome-free serum, 100 U/mL of penicillin, 100 μg/mL streptomycin, and 9.0 g/L glucose at 37 °C and 5% CO2. Collect the conditioned medium after a 48 h culture and proceed to the isolation of EVs….
Açıklamalar
The authors have nothing to disclose.
Materials
| Rat Macrophage & Microglia Culture Medium | Cell Applications | R620K-100 | Cell orgin: Normal healthy Rat bone marrow |
| Rat primary microglia | Lonza | RG535 | |
| Sepharose CL-2B | GE Healthcare | 17014001 | |
| Ultracentrifuge | Beckman Coulter | A95765 | |
| Ultracentrifuge Rotor 70.1 Ti | Beckman Coulter | 342184 | |
| Glucose | Sigma-Aldrich | G8769 | |
| Phosphate Buffer Saline | Invitrogen | 14190094 | No calcium, No magnesium |
| Polycarbonate centrifuge tubes | Beckman Coulter | 355651 | |
| Penicillin-Streptomycin | Gibco | 15140-122 | |
| Exo-FBS | Ozyme | EXO-FBS-50A-1 | Exosome depleted FBS |
| DMEM Gibco 41966029 | |||
| Centrifuge | Eppendorf | 5804000010 | |
| CO2 Incubator | ThermoFisher | ||
| pluriStrainer M/ 60 μm | pluriSelect | 43-50060 | |
| MonoP FPLC column | GE Healthcare | No longer available |
Etiketler
Bu Makaleden Alıntı Yapın
SEC-based Isolation of Microglia-derived Extracellular Vesicles: A Technique to Isolate Extracellular Vesicles from Microglia Conditioned Culture Media. J. Vis. Exp. (Pending Publication), e20623, doi: (2023).