Culturing DRGs
References:
A dissection and Tissue Culture Manual of the Nervous System (1989). A. Shahar, J.D. Vellis, A. Vernadakis, B. Haber (Eds.), Dissociated Spinal Cord - Dorsal Root Ganglion Cultures on Plastic Tissue Culture Dishes and Glass Coverslips and Wells (pp.219-222). Wiley-Liss, Inc.
J.L. Werth, S.A. Thayer (1994) Mitrochondria Buffer Physiological Calcium Loads in Cultured Rat Dorsal Root Ganglion Neurons, The Journal of Neuroscience, 14(1), 348-356.
Materials
Collagenase/Dispase (Roche 10269638001). Dissolve at 100 mg/ml water; dilute 1:100 with Hibernate A-Ca( BrainBits, LLC Cat#: HA-Ca) to 1 mg/ml = 0.8U Collagenase/6.4U Dispase.
Sterile filter, aliquot and freeze (1 ml aliquots). Thaw aliquot on day of use.
Poly-D-Lysine (0.15ml/cm2 substrate, 50 ug/ml water, 135 kD, 1-20 hr. e.g. Sigma P4607 store at -20o C)
Calf Skin Collagen (1:12 dilution in sterile 18 ohm water, from stock in 0.1 M acetic acid overnight e.g. Sigma C8919 store -20o C).
Dissociation Medium: Hibernate A-Ca + B27 + Glutamax
Plating Medium: 12 ml NbActiv4 + NGF (dilute 50ug/ml stock 1:2000 = 25ng/ml)(BrainBits, LLC Cat# NbActiv4 supplied) or Neurobasal/B27/Gluamax + NGF (dilute 50ug/ml stock 1:2000 = 25ng/ml) (components available at Invitrogen)
Substrate:
15mm round glass coverslips (Carolina Biological Supply #63-3031). Sterilize by autoclaving.
Place 3 slips in a sterile 35mm perti dish (VWR# 25382-064). Coat the Substrate with one of the following: Neurons will attach better to PDL and grow fibers better.
A) Poly-D-Lysine (100ug/ml water store at -20o C ).Aliquot 200 ul to substrate and allow to sit for at least 1 hour. Rinse once and allow to dry.
B) Aliquot 200 ul of Calf Skin Collagen (1:12 dilution in sterile 18 ohm water, from stock in 0.1 M acetic acid store at -20o C) on each slip. Allow to dry overnight. Next day aspirate off any excess collagen and let dry (DO NOT RINSE).
DRG Dissociation
1. Transfer the ganglia with minimum Hibernate E+ B27+ Glutamax, into 1 ml Dissociation medium containing 1 mg/ml Collagenase(0.8U)/Dispase(6.4) ( HA-Ca + Glutamax + 0.25% Collogenase/Dispase) in a 50ml centrifuge tube.
2. Incubate for 30 minutes at 30oC with shaking every 5 min.
3. Allow tissue to settle. Then transfer tissue with minimal protease to a 15ml centrifuge tube that contains 3ml room temperature Hibernate A-Ca + B27 + Glutamax (BrainBits # HA-Ca, B27 and Glutamax available from Invitrogen).
4. Dissociate by trituration with sterile glass fire-polished pipette up to 30 times or until 90% single cell suspension. If not possible, incubate up to 30 min longer at 30oC.
5. Let the undissociated pieces settle for 1 minute.
6. Transfer supernatant containing cells into a 15ml Centrifuge tube.
7. Centrifuge cell suspension 1 min at 1100 rpm (200 x G) to collect cells.
8. Remove supernatant.
9. Resuspend pellet in 2ml NbActiv4+ NGF
10.Aliquot 20ul and mix with 20ul of 0.4% trypan blue (1:1 dilution). Count in hemacymtometer.
11. Dilute the DRG neurons for plating to 105 cells/cm2 substrate (15mm diameter slips are 1.77cm2).
12. Incubate cultures in a humidified incubator at 37oC, 5% CO2, 9% O2 (Optional: 37oC, 5% CO2, ambient O2).
13. After 1 hour, carefully flood dish with 2 ml 37oC / 5% CO2 - equilibrated plating media.