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1. Treatment of Db-Camp and Collection of Macrophage Conditioned Medium
NOTE: Start db-cAMP treatment 4 h after the neuron-macrophage co-cultures.
- Add 2 µL of 100 µM dibutyryl cyclic AMP (db-cAMP) solution to the neuron-macrophage co-cultures. Add the same volume of phosphate-buffered salin (PBS) for a control experiment.
- After 24 h, fill an empty well with 1 mL of macrophage culture medium in the same 6-well plate. Transfer the cell culture insert in the neuron-macrophage co-cultures to the empty well with macrophage culture medium. Keep the cells under the same condition for 72 h without changing the medium.
NOTE: We add only 1 mL of macrophage culture medium during conditioned medium (CM) collection to make concentrated CM. During the CM collection, if needed, we added more to completely cover the macrophages within the insert.
- After 72 h, centrifuge the macrophage CM at 239 x g for 5 min to remove the cellular components. Pass the supernatant through a 0.2-µm filter to remove any remaining cellular debris. Store the collected CM at -70 °C until use.
2. Neurite Outgrowth Assay with Collected CM
- Before setting up a separate adult DRG neuron culture, pre-coat an 8-well chamber slide 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 temperature at least for 1 h.
- Obtain dissociated adult DRG neurons in Neurobasal medium supplemented with B27. Plate 5 x 104 cells per well onto the pre-coated 8-well chamber slide.
- Place the chamber slide in a 37 °C incubator for 2 h, allowing the cells to attach to the bottom. Then, replace the culture medium with the thawed CM that is preheated at 37 °C.
NOTE: Be careful not to touch the bottom of the 8-well chamber slide when removing the culture medium and adding the collected CM.
- 15 h after the initial plating, remove the medium and wash the cells with PBS once. Then, add 200 µL of ice-cold 4% paraformaldehyde solution to the wells, and incubate the cells with the paraformaldehyde solution for 20 min at 4 °C
NOTE: The DRG neuron culture for neurite outgrowth assay should be precisely restricted to 15 h. Under this condition, there is no appreciable neurite outgrowth.
- Wash three times with ice-cold PBS and then perform blocking with 10% normal goat serum (NGS) with 0.1% Triton-X for 30 min. Incubate the fixed cells with primary antibody (anti-Tuj-1) solution diluted with 10% NGS (at a concentration of 1 µg/mL) for 4 h at room temperature or overnight at 4 °C.
- Wash three times with PBS, and then incubate the cells with secondary antibody (goat-anti mouse) solution for 2 h at room temperature. Then, wash twice with PBS and mount the culture slide with a coverslip (24 × 50 mm) using a mounting solution.
- Take images using a fluorescence microscope to visualize neurite outgrowth (Figure 1).
