Developing a Neuron and Macrophage Coculture In Vitro

Abstract

Source:Yun, H. J., et al. Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity. J. Vis. Exp. (2018).

This video shows the co-culturing of murine dorsal root ganglion neurons and macrophages. Neurons are seeded on a coated plate, while macrophages are collected from an euthanized mouse, treated, and added to the culture insert. Treatment with db-cAMP activates neurons and prompts macrophages to adopt a pro-regenerative phenotype to support neuronal outgrowth.

Protocol

1. Culture Preparation of Dissociated Adult dorsal root ganglion Neuron or DRG Neuron Before setting up the culture, pre-coat a 6-well plate with poly-D-lysine and laminin. Incubate a 6-well plate with 0.01% poly-D-lysine at 37°C for 2 h or at 4°C overnight. Then, wash the plate twice with distilled water. Incubate the plate with laminin solution at a concentration of 3 µg/mL for 2 h at room temperature, and then wash the plate twice with distilled water. Dry the plate at room tempera…

Materials

Cell culture insert transparent PET membrane 0.4μm pore size	Corning,Falcon	353090	Transparent PET membrane with 0.4-μm pore size, for 6-well plate
70-μm nylon cell strainer	Corning, Falcon	352350
Red blood cell lysis buffer	Qiagen	158904
Neurobasal medium	Thermo Fisher Scientific, Gibco	21103-049	Containing 1% glutamax and 1% penicilin-streptomycin
Poly-D-lysine Hydrobromide	Sigma-Aldrich	P6407-5MG
Laminin Thermo Fisher Scientific,	Invitrogen	23017-015
Adenosine 3', 5'-cyclic monophosphate, N6 ,O2'-dibutyryl-, sodium salt	Merck Millipore Corporation, Calbiochem	28745
Cell culture CO2  incubator	Panasonic	N/A