Isolation of Neurons from Caenorhabditis elegans via Fluorescence-Activated Cell Sorting

1. Preparation and collection of aged worms for cell isolation

1. Collect worms and prepare buffers for cell isolation.
   1. Collect worms in a 15 mL conical tube. Wash worms from plate with 1.5 mL of M9 buffer (1 mM MgSO₄, 85 mM NaCl, 42 mM Na₂HPO₄·7H₂O, 22 mM KH₂PO₄, pH 7.0) and centrifuge for 5 min at 1,600 x g. Discard supernatant and wash worms with 1 mL of M9 buffer. Repeat centrifugation and wash for a total of five times to remove as much Escherichia coli contamination as possible.
      ​NOTE: Adding antibiotics (50 µg/mL), such as ampicillin, at this step can help reduce bacterial contamination.
   2. For two samples, prepare 2 mL of sodium dodecyl sulfate-dithiothreitol (SDS-DTT) lysis buffer: 200 mM DTT, 0.25% (w/v) SDS, 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (pH 8.0), and 3% (w/v) sucrose.
   3. Prepare 15 mL of isolation buffer (118 mM NaCl, 48 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 25 mM HEPES [pH 7.3]) and store it on ice.
      NOTE: Both the SDS-DTT lysis buffer and isolation buffer must be made fresh before each experiment.
2. Cuticle disruption and single cell isolation
   1. Centrifuge animals collected in step 1.1.1 for 5 min at 1,600 x g. Remove all supernatant and suspend worms in 1 mL of M9 media and transfer to 1.5 mL microcentrifuge tube.
   2. Pellet worms with centrifugation at 1,600 x g for 5 min.
   3. Add 200 µL of SDS-DTT lysis buffer to worms and incubate for 5 min at room temperature (RT). Worms should appear to be "winkled" along the body if viewed under a light microscope.
      NOTE: Prolonged exposure to SDS-DTT lysis buffer may result in death of worms, which can be monitored by observing worms. Worms that are dead will elongate and will not curl.
   4. Add 800 µL of ice-cold isolation buffer and mix by gently flicking tube.
   5. Pellet worms for 1 min at 13,000 x g at 4 °C, remove supernatant and wash with 1 mL of isolation buffer.
   6. Repeat step 1.3.5 for a total of five times, carefully removing isolation buffer each time.
   7. Add 100 µL of protease mixture from Streptomyces griseus (15 mg/mL) (Table of Materials) dissolved in isolation buffer to the pellet and incubate for 10−15 min at RT.
      NOTE: As with the SDS-DTT lysis buffer, the extended protease digestion may result in excessive cleavage of proteins along the plasma membrane, preventing isolation of surface exposed green fluorescent protein (GFP) via magnetic beads.
   8. During incubation with protease mixture, apply mechanical disruption by pipetting samples up and down against the bottom of the 1.5 mL microcentrifuge tube with a 200 µL micropipette tip for ~60−70 times. Keep the pipette tip against the wall of the microcentrifuge tube with constant pressure to properly disassociate cells.
   9. To determine the stage of digestion, remove a small volume (~1−5 µL) of the digestion mixture, drop it on a glass slide and inspect it using a tissue culture microscope. After 5−7 min of incubation, worm fragments should have visibly reduced cuticle and a slurry of cells will be readily visible.
   10. Halt reaction with 900 μL of commercially available cold Leibovitz's L-15 medium supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (final concentration at 50 U/mL penicillin and 50 μg/mL streptomycin).
   11. Pellet isolated fragments and cells by centrifugation for 5 min at 10,000 x g at 4 °C. Wash the pelleted cells with 1 mL of cold L-15-supplemented media twice more to ensure that excess debris and cuticle is removed.
   12. Resuspend pelleted cells in 1 mL of L-15-supplemented media and leave on ice for 30 min. Take the top layer, approximately 700−800 µL, to a microcentrifuge tube. This layer contains cells without cell debris and will be used in the subsequent isolation of cells of interest.
   13. Following manufacturer's instructions, use an automated cell counter or hemocytometer to measure the cell density of 10−25 µL of isolated cells.

2. Isolation of GFP-positive cells via flow cytometry

1. Isolation of GFP-positive cells by flow cytometry
   1. From isolated cell suspension collected in step 1.2.12, centrifuge cells for 5 min at 10,000 x g at 4 °C. Discard supernatant and suspend pelleted cells in L-15-supplemented medium to a cell density of no more than 6 x 10⁶ cells/mL to avoid overloading the flow cytometer.
   2. Sort cells by GFP-positive expression using a flow cytometer capable of sorting samples. GFP-negative cells can be used as a control for non-cell type specific analysis.
2. Culturing of isolated cells
   1. Plate isolated cells into a sterile 6-well multi-well plate.
      NOTE: These plates may be coated with peanut lectin to increase cell attachment; however, if cells are to be used immediately, this step may be disregarded.
   2. Place the plate into a plastic container and incubate cells at 20 °C with a damp wipe or soft tissue paper (add 50 µg/mL ampicillin to double distilled water [ddH₂O] to ensure no bacterial growth on wipe) to add moisture to cells. Do not incubate in a CO₂ incubator, as elevated CO₂ levels may damage the cells.