Method Article

SEC-based Isolation of Microglia-derived Extracellular Vesicles: A Technique to Isolate Extracellular Vesicles from Microglia Conditioned Culture Media

April 30th, 2023

In This Article

Abstract

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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

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1. Primary Culture of Microglia/Macrophages

  1. Primary culture of microglia
    1. 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.
    2. Collect the conditioned medium after a 48 h culture and proceed to the isolation of EVs.
  2. Primary culture of macrophages
    1. Culture commercial rat primary macrophages (1 x 106 cells) in the medium pr....

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Disclosures

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No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Rat Macrophage & Microglia Culture MediumCell ApplicationsR620K-100Cell origin: Normal healthy Rat
bone marrow
Rat primary microgliaLonzaRG535
Sepharose CL-2BGE Healthcare17014001
Ultracentrifuge Beckman Coulter A95765
Ultracentrifuge Rotor 70.1 Ti Beckman Coulter 342184
Glucose Sigma-Aldrich G8769
Phosphate Buffer Saline Invitrogen 14190094No calcium, No magnesium
Polycarbonate centrifuge tubesBeckman Coulter 355651
Penicillin-Streptomycin Gibco 15140-122
Exo-FBS Ozyme EXO-FBS-50A-1 Exosome depleted FBS
DMEM Gibco 41966029
Centrifuge Eppendorf 5804000010
CO2 IncubatorThermoFisher
pluriStrainer M/ 60 μm pluriSelect 43-50060
MonoP FPLC column GE Healthcare No longer available

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Tags

Extracellular VesiclesMicroglia EVsSize Exclusion ChromatographyEV IsolationMicroglia Conditioned MediaUltracentrifugationGel FiltrationEV ElutionProtein AggregatesEV Fractions

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