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

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….

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