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

Protocol

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.

Materials

Name	Company	Catalog Number	Comments
6-well plate	Fisher Scientific	12-556-004
Agar, Molecular Biology Grade	VWR	A0930
CaCl2	Sigma	C5670
Contess Automated Cell Counter	Invitrogen	Z359629
DTT	USBiological	D8070
FBS	Omega Scientific	FB-02
HEPES	Sigma	H3375
KCl	Amresco	O395
KH2PO4	USBiological	P5110
Kimwipes	Kimberly-Clark Professionals	7552
Leibovitz's L-15 Medium	Gibco	21083027
MgCl2	Sigma	M8266
MgSO4	USBiological	M2090
Na2HPO4·7H2O	USBiological	S5199
NaCl	VWR	X190
NaOCl (Bleach)	Clorox
NaOH	Amresco	O583
Penicillin-Streptomicen	Fisher Scientific	15140122
Peptone Y	USBiological	P3306
Pronase E	Sigma	7433	protease mixture from Streptomyces griseus
SDS	Amresco	O227
SMT1-FLQC fluorescence stereomicroscope	Tritech Research
Sucrose	USBiological	S8010