Neuronal cultures

E12.5 DRGs were isolated from mouse spinal cords. We roughly have 600k cells/embryo (20k cells/spots; ~30 spots/embryo). 

For STORM imaging experiments, cultures were plated on poly-D-lysine (40 µg/ml, P6407-5mg), and then with substrate solution containing N-cadherin (1 µg/ml, R&D 1388-NC) and fibronectin (5 µg/ml, R&D 1918-FN). All other cultures were plated on poly-D-lysine (100 µg/ml) and mouse natural laminin (10 µg/ml, Life Technologies).

Coating

1. Apply 300ul poly-D-lysine (40 µg/ml) in water to each well of four 4-well slider
2. Put in 37C incubator overnight.
3. Wash wells with water once
4. Add 200ul substrate solution to each well, shake to cover corners
5. Incubate for >2h <12h in 37C incubator.

Cell culture

1. Dissociate DRGs in 500ul 0.05% Trypsin-EDTA in PBS (Life Technologies) for 20 min; flick at 10min to enhance dissociation. 
2. Quench with 500ul 10%FBS/DMEM (no antibiotics) without taking out Trypsin
3. Spin at 500g for 2.5min
4. Aspirate then resuspend in 1ml of 10%FBS/DMEM
5. Triturate with P1000 (avoid bubbles), ~15x up and down
6. Remove with p20 any un-dissociated piece
7. Spin 500g, 4.5min to pallet
8. Resuspend in 20ul * number of embryo in culture media: Neurobasal (Life Technologies) containing 2% (v/v) B27 (Life Technologies), 0.45% (v/v) Glucose, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and supplemented with 50 ng/mL NGF (Promega).
9. Count cell density 
10. Resuspended Cells were plated on dried substrate in multi-well chamber slides at a density of ~15,000 per 1–1.5 microliters, allowed to adhere in a humidified incubator for ~12–15 min
11.  Confirm cell adherence under the microscope. 
12.  Fill the chambers with media (600ul medium from the corner of 4-well slider) and cultures returned to the incubator to continue culturing at 5% CO2 and 37C. 

The plating configuration allows for radial axon outgrowth away from a pool of cell bodies located near the center of the well. For imaging proximal axons, we used dissociated cells uniformly plated at a reduced density of ~4000 per well to avoid axon bundling. 

Post-plating day 1:

1. Prepare 100*# of wells ul  culture medium with mitotic inhibitor for later 1:6 dilution (for example, a final concentration of 5 µM 5-fluorouracil and 5 µM uridine). 
2. Add 100ul to each well. 

Post-plating day 3 and 5

1. Prepare 350* # of wells ul culture medium
2. Put in incubator for ~3h with lose cap to equilibrate
3. Take out 250ul per well and add 350ul per well

All assays were performed at 7–8 days in vitro (DIV).