Ganglia dissociation and single-cell sorting

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

Carefully remove fat and connective tissue from the SG, and then transfer the SG to a new dish containing cold equilibrated EBSS

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)

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)

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

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

Invert the tubes a few times gently and centrifuge them at 300g at 4C for 5 min

Discard the supernatant and gently resuspend the pellet with 500 μl of the digestion-stop solution described above

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

We progressively decreased the diameter of the pipettes from 300-400 μm to 150 μm during the trituration process

The contents were then divided again into two tubes and washed with 1 ml of the medium solution

We inverted the tube gently 10 times and centrifuged it at 300 g for 5 min

The supernatant was discarded and the pellets were gently re-suspended with 200 μl of the medium solution

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

Keep the cell suspensions on ice until sorting

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

Centrifugate the plates at 200g for 2 min and immediately freeze them on dry ice