DRG cultures

Kalinski Lab- Ball State University: Adult Mouse DRG culturing protocol:

Complete Media

10% Fetal Bovine Serum (I prefer Neuromics, but Gibco is fine)

1X N1 Supplement (supplied as 100x, Sigma) or 1X N2 Supplement (100x stock)

DMEM F-12 50/50 Mix w/glutamax

Wash Media

10% Fetal Bovine Serum

1x PenStrep (P/S supplied as a 200x stock)

DMEM F-12 50/50 Mix w/glutamax

PenStrep Wash

1x PenStrep

1X PBS (cell culture grade PBS)

PenStrep/L-15 Wash

1x PenStrep

L-15 Media

Transport Buffer

1 mL of PenStrep/L-15 Wadh

Preparing plates:

1. Apply poly-l-lysine (PLL) (P8920 Sigma) to plates leave 30-45 minutes in 37 degree incubator.
2. Remove PLL (you can reuse this a dozen times or so), and leave in hood to dry for about 1 hour.
3. Rinse once with dH20
4. Apply laminin (mixed with PBS) 2-20 ug/mL. Adjust based on density of culture (lower laminin concentration for higher density culture). This is a dilution of 1:100 to 1:500. 
5. Let plates rock in 4 degree for 1-2 days, or 2 hours at 37 degrees.

Culture:

1. Harvest adult DRGs and put in transport buffer on ice during dissection. (be sure to trim the DRGs as much as possible to help in the digestion step)
2. Fill 5 wells of a 24 well plate with 0.5mL of PenStrep/L-15. Rinse DRGs in each well then place in a well with 400ul of DMEM F-12 50/50.
3. Chop DRGs with microscissors to start the breakdown of tissue
4. Add 50 ul of 10mg/ml Collagenase and 50 ul of 10mg/ml dispase (for digestion I limit to 12 DRGs, you can pool the DRGS for wash steps after digestion)
5. Leave in incubator for 20 minutes, triturate with fire polished glass pipette about 20 times. Can leave in incubator an additional 5-20 if needed. 
6. Triturate another 15 times then transfer to a 15 mL conical tube
7. Add up to 12 mL Wash Media and spin for 5 minutes at 160-200 x g in swinging bucket centrifuge (make sure it is balanced) at room temperature. 
8. Aspirate media and resuspend pellet in 1 mL wash media. Triturate about 10 times, then fill up to 12 mL media. Repeat spin.
9. Aspirate media and resuspend pellet in complete media. Plate as desired. 
   1. I usually plate 0.25-1 DRG per well of a 24 well plate. 0.25 DRGs if you want to analyze axons and up to 1 DRG if you want to measure neurite outgrowth. 
   2. If you want to measure outgrowth limit culture time to 18 - 22 hours.