Adult rat DRG neuron culture

Procedure 

Day 1: 1. Surgical instruments (scissors and forceps) are sterilized with autoclaving, followed by drying using the oven-drying method, and then stored at 4 °C. 

2. A 24-well plate with cover glasses is coated with Poly-L-lysine (PLL, Sigma, P4832) at 4 °C overnight. 

Day 2: 

1. Remove the PLL solution from the 24-well plate containing cover glasses, and rinse the cell plate three times with sterile deionized water. The plate is left open to dry in a laminar flow hood for 30 minutes. 

2. A 35 mm Petri dish is added with pre-cooled Hibernate-A medium (Gibco, A1247501) and placed on ice. 

3. The rat is sacrificed via CO₂ inhalation. The skin surface is disinfected with iodophor after shaving. The skin is incised at the ventral midline using standard scissors. The spinal cord is rapidly dissected with a pair of standard scissors. Two long cuts are made closely to the left and right of the spinal column. The tail and skull are removed. Finally, the remaining paravertebral muscles are largely removed to expose the spinal column. Remove the entire spine using hemostatic forceps and large scissors. 

4. Open the spine, remove the spinal cord, expose the intervertebral foramen, and carefully extract the DRGs with microforceps. DRGs from all spinal levels are carefully isolated and collected in a 35-mm Petri dish filled with pre-cooled Hibernate-A medium placed on ice. Approximately 30 to 40 DRGs are obtained based on the experimenter's expertise. 

5. Collagenase solution (3 mg/mL, Roche, 11088793001) is prepared and preheated for 10 minutes at 37°C. 

6. The DRGs were transferred to a sterile 5 mL centrifuge tube, and the Hibernate-A medium was discarded. DRGs are rinsed with pre-cooled sterile PBS until the blood on the surface is removed. 

7. 1 mL of preheated collagenase was added, and the DRG tissue was cut with sterile microscissors. 

8. The cut DRGs were transferred to a sterile petri dish, supplemented with preheated collagenase up to 3 mL, and digested in a CO₂ incubator for 90 minutes. 

9. An aliquot of the 0.25% tyrisin (Gibco, 15050065) solution is thawed and preheated for 20 minutes at 37 °C. 

10. After digesting with collagenase for 90 minutes, the suspension was aspirated using a 1,000 µL pipette, and 3 mL of preheated 0.25% trypsin was added to it. The mixture was then placed in a CO₂ incubator for further digestion. 

11. During digestion, the suspension was agitated repeatedly with a 1000 µL pipette every 5 minutes to ensure complete digestion (no visible tissue mass). Then, 2 times the volume of preheated DMEM (Gibco, 11965092) with 10% FBS (Sigma, F8687) was added to the medium to stop the digestion process, followed by centrifugation at 1000 rpm for 5 minutes at room temperature. 

12. Discard the supernatant, add 6-8 mL of preheated 15% bovine serum albumin (BSA, Sigma, A1933) for purification. Resuspend the cells and centrifuge at 900 rpm for 5 minutes at room temperature. 

13. Carefully discard the supernatant and impurities using a 1,000 µL pipette. Add 6-8 mL of preheated 15% BSA, resuspend the cells, filter them through a 70 μm nylon mesh cell strainer (BD, 352350), and centrifuge at 900 rpm for 5 minutes at room temperature. 

14. The supernatant is removed from the cell pellet using a 1,000 µL pipette. An appropriate amount of Neurobasal medium (Gibco, 10888022) with 2% B-27 Supplement (Gibco, 17504044) and 1% GlutaMax (Thermo Fisher Scientific, 35050087) is added for re-suspension. The cells are then plated in the PLL-coated cell plate from step 1.