Isolation of Cells from Sensory Ganglia

All procedures involving sample collection have been performed in accordance with the institute’s IRB guidelines.

1. Sensory Ganglia Dissociation

1. Pipette out 500 µl Ganglia Dissociation Solution (GaDS) (Table 1), leaving ganglia in the tube. Wash ganglia once by adding 1 ml Phosphate buffer solution (PBS), wait for ganglia to settle to the bottom of the tube, and then pipette out PBS.
2. Add 1 ml GaDS and 22 µl of digestion enzyme (collagenase I/II and protease mix, 2.5 mg/ml in water) to each tube.
3. Add 20 µl Fast Blue (5.26 mM) to the positive control tube.
4. Put the tubes in a water bath or heating block set at 37 °C.
5. Every 15 min, shake the tubes briefly and flick them to ensure the ganglia are covered by the solution.
   Note: The time of ganglia digestion depends on the age of the digestion enzyme. Within one month of dissolving the digestion enzyme, digest for 30 min; within 2 – 6 months, digest for 45 min. If the enzyme is over six months old, digest the ganglia for 60 min.
6. While ganglia are being digested, prepare density gradients using a particle solution (colloidal silica particles coated with polyvinylpyrrolidone).
7. Mix 12% particle solution in GaDS, 500 µl per sample.
8. Mix 28% particle solution in GaDS, 500 µl per sample.
9. Slowly and carefully add 400 µl 12% density solution on top of 400 µl 28% density solution in a fresh 1.5 ml tube for each sample. Do not allow the two layers to mix. Two distinct layers should be visible in the tube, one darker than the other.
10. After the appropriate digestion time, remove GaDS with digestion enzyme and wash ganglia twice with 1 ml PBS. Add 200 µl of fresh GaDS to each tube.
11. Use a 200 µl pipette, set to 100 µl volume, and pipette ganglia up and down several times to break cells apart. Repeat until no intact piece of tissue is seen. Avoid creating bubbles in the solution.
12. Pass dissociated cells through a 70 µm cell strainer.
13. Add another 100 µl GaDS to the original dissociation tube to pick up any remaining stray cells left and pass the additional solution through the cell strainer. Using a new pipette tip, collect any remaining cell-containing liquid that has passed through the strainer and clung to the mesh. The final volume of cells suspended in GaDS is 300 µl.
14. Carefully layer the 300 µl of cells on top of the previously made density gradient. Avoid bubbles and mixing of cells with the density layers.
15. Centrifuge for 10 min at 2,900 x g at RT.
16. Once centrifugation is complete, carefully remove and discard the top 700 µl layer. This layer contains the majority of cell debris that will otherwise interfere with cell sorting.
17. Add 700 µl fresh GaDS to the remaining cell containing solution and pipette up and down several times to mix.
18. 18. Centrifuge for 15 min at 2,900 x g to pellet cells.
19. 19. Once centrifugation is done, carefully remove and discard the supernatant, leaving the cell pellet.
20. Resuspend the cell pellet in 200 – 300 µl GaDS by pipetting up and down with a 1,000 µl pipette set to 200 µl several times. Put samples on ice.

Table 1. Reagent Mixture for Ganglia Dissociation Solution (GaDS).