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 |