TABLE 1.
TROUBLESHOOTING
| Step | Problem | Possible cause | Solution |
|---|---|---|---|
| Equipment setup | Few neurons found on coverslip | Coverslips are not properly coated; bad or expired PDK/collagen | Verify that PDK/collagen was made properly and that it did not spill off coverslip for entire duration of coating procedure |
| 15–23 | Over-trituration; too much enzymatic digestion | Triturate with larger tip diameter pipette; triturate for less time; Decrease enzyme incubation times. | |
| 15–23 | Under-trituration; not enough enzymatic digestion | Triturate with a smaller tip diameter pipette and/or for longer; cut tissue into smaller pieces; increase enzyme incubation times. | |
| 15–33 | Unhealthy cells due to prolonged dissociation | Expedite culturing procedure and reduce time outside incubator | |
| 4–6 | Anterior spinal column cannot be removed | Vertebral bodies or pedicles are not fully transected, there are remaining bone or connective tissue attachments | Apply greater force when using osteotomes and mallet to ensure bone connecting to the anterior spinal column is fully detached |
| 6–7 | Cannot localize DRG after spinal column is removed and dura is torn | Tearing due to the pulling of dura and DRG by the posterior longitudinal ligament | Take more time to ensure dura is completely detached from the posterior longitudinal ligament during column removal; check posterior portion of spinal column for DRG that may be attached |
| 24 | Cell strainer clogs, few cells in suspension after filtration | Too much debris has blocked dissociated cells from passing through cell strainer | Cut tissue into smaller pieces, use more than one cell strainer to filter less volume at one time |
| 31–32 | Cell suspension spills off of coverslips into well bottom during plating | Coverslips are not dry prior to plating | Dry coverslips in uncovered dish in tissue culture hood for longer period of time prior to cell plating; vacuum several times to remove any remnants of PDK/collagen solution (see Equipment setup). |
| 34 | Bacterial/fungal contamination | Contamination of cells during dissection, trituration, or incubation | Perform dissection under sterile conditions (blow out hood, BSL2 tissue culture hood) using sterilized tools; ensure penicillin/streptomycin is fresh; clean and sterilize incubator |