Jul 06, 2022

Public workspaceGanglia dissociation and single-cell sorting

DOI
dx.doi.org/10.17504/protocols.io.14egn79e6v5d/v1
  • 1Columbia University Irving Medical Center
Daniele Neri
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DOI: dx.doi.org/10.17504/protocols.io.14egn79e6v5d/v1
Protocol Citation: Seoeun Lee, Daniele Neri, Lori Zeltser 2022. Ganglia dissociation and single-cell sorting. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn79e6v5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: April 01, 2022
Last Modified: July 06, 2022
Protocol Integer ID: 60199
Abstract
Protocol to dissociate freshly harvested stellate ganglia into single neurons and to sort them based on fluorescence
Materials

ABC
InstrumentProviderCat. no/SKU
40 µm Cell StrainerCorning352340

ABC
ReagentProviderCat. no/SKU
Earle's Balnced Salt SolutionThermofisher Scientific14155063
PapainWorthington BiochemicalLK003178
Collagenase/DispaseRoche11097113001
D-trehaloseSigma-Aldrich90208
AP-VTocris0105
kynurenic acidSigma-AldrichK-3375
DNAseWorthington BiochemicalLK003170
Fetal Bovine Serum, certified, heat inactivatedFisher Scientific10-082-147
DMEMFisher Scientific11995073
Bovine Serum AlbuminSigmaA2153-50G
RNAse-Free WaterFIsherScientificBP5611
Sytox blueThermofisher ScientificS34857

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)
Cleaning the tissue
Cleaning the tissue
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
Digestion
Digestion
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
Dissociation
Dissociation
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
Staining and preparation for sorting
Staining and preparation for sorting
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
Sorting
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