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…
Divulgazioni
The authors have nothing to disclose.
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