TITLE: Ganglia dissociation and single-cell sorting
AUTHORS: Seoeun  Lee, Daniele  Neri, Lori  Zeltser

[DESCRIPTION]
Protocol to dissociate freshly harvested stellate ganglia into single neurons and to sort them based on fluorescence

[BEFORE_START]
We modified the dissociation methods from protocols previously published (Saxena et al., 2012, https://doi.org/10.2144/0000113878; Campbell et al., 2017, https://doi.org/10.1038/nn.4495)

[STEPS]
SECTION: Cleaning the tissue
1. Place a freshly harvested Stellate Ganglion (SG) into an ice-chilled Earle’s Balanced Salt Solution (EBSS) that was equilibrated to 95% CO2/ 5% O2 for 1 hour
SECTION: Cleaning the tissue
2. Carefully remove fat and connective tissue from the SG, and then transfer the SG to a new dish containing cold equilibrated EBSS
SECTION: Digestion
3. Cut the SG into 3-4 pieces using a small spring scissor, and placed all pieces gently into a low-bind 1.7 ml microcentrifuge tube containing 1,667 μl pre-heated (37°C) digestion solution for 1.5 h with constant agitation
Digestion solution is composed of:
1034 μl Papain solution  in EBSS
200 μl Collagenase/Dispase solution (20 mg/ml in EBSS)
167 μl D-trehalose solution (50% in RNase-free water)
3 μl AP-V solution (25mM in EBSS)
13 μl kynurenic acid solution (100mM in EBSS)
250 μl DNAse (vial D2 in EBSS)
SECTION: Digestion
4. During the digestion, prepare a digestion-stop solution:
1,050 μl 50% D-trehalose solution
11 μl 25 mM AP-V solution
44 μl of the 100 mM kynurenic acid,
250 μl of the DNAse solution
250 μl fetal bovine serum (FBS)
SECTION: Digestion
5. Prepare medium solution containing 
1,050 μl 50% trehalose solution
8 μl the 25 mM AP-V
19ul 100 mM kynurenic acid
107 μl FBS in 9,450 μl D-MEM/F12
SECTION: Digestion
6. After the digestion, transfer half of the digestion solution with the tissue to a fresh low-bind microcentrifuge tube, and fill each tube with the digestion-stop solution
SECTION: Dissociation
7. Invert the tubes a few times gently and centrifuge them at 300g at 4C for 5 min
SECTION: Dissociation
8. Discard the supernatant and gently resuspend the pellet with 500 μl of the digestion-stop solution described above
SECTION: Dissociation
9. We combined the contents into a single tube and triturated the SG carefully with fire-polished glass Pasteur pipettes that were pre-coated with 0.5% BSA in RNase-free water for at least 1h at room temperature
SECTION: Dissociation
10. We progressively decreased the diameter of the pipettes from 300-400 μm to 150 μm during the trituration process
SECTION: Dissociation
11. The contents were then divided again into two tubes and washed with 1 ml of the medium solution
SECTION: Dissociation
12. We inverted the tube gently 10 times and centrifuged it at 300 g for 5 min
SECTION: Dissociation
13. The supernatant was discarded and the pellets were gently re-suspended with 200 μl of the medium solution
SECTION: Staining and preparation for sorting
14. The suspension was filtered using a 40 μm cell strainer and collected in a 15-ml plastic tube. We used Sytox blue (1:1000) to stain dead cells
SECTION: Staining and preparation for sorting
15. Keep the cell suspensions on ice until sorting
SECTION: Sorting
16. Sort single cells into 96-well plates, We used FACSAria sorter with a square cuvette with a 130-μm nozzle at 12 PSI.
We sorted based on the the signal from Alexa 488, Alexa 555, or the lack of any signal
SECTION: Sorting
17. Centrifugate the plates at 200g for 2 min and immediately freeze them on dry ice