TITLE: Synchronized C. elegans culture on NGM plates for FACS isolation of intestine cells
AUTHORS: Robert TP Williams, Erin Osborne Nishimura

[DESCRIPTION]
This protocol details the steps necessary to scale-up and synchronize C. elegans cultures for FACS isolation of intestine cells. We cultured worms with agar-based NGM plates to reduce any confounding effects that may be introduced by large scale liquid culture. This protocol utilizes two rounds of mixed stage culture growth followed by two rounds of synchronized growth. After scale-up and synchronization, this protocol provides details for culture conditions necessary for intestine FACS of embryo, L1 or L3 stage experiments.

[STEPS]
SECTION: Prepare OP50 seeded NGM plates
1. Using sterile technique, pick an OP50 colony and inoculate a 250 ml bottle of sterile LB.
SECTION: Prepare OP50 seeded NGM plates
2. Incubate OP50 liquid culture at 37°C overnight
SECTION: Prepare OP50 seeded NGM plates
3. Make 3 liters of Nematode Growth Media (NGM) with 150 mm petri dish (hereafter referred to as "large plates"). See the following WormBook page for NGM protocol.
SECTION: Prepare OP50 seeded NGM plates
3.1. For FACS isolation of post-embryonic worm stages, prepare an additional 1 liter of peptone enriched NGM media. For peptone enriched NGM media, replace the normal 2.5 g peptone mass with 20 g. 
SECTION: Prepare OP50 seeded NGM plates
4. Pour molten NGM into large plates. Each liter should make 20 plates, for a total of 60 large plates
SECTION: Prepare OP50 seeded NGM plates
5. Allow plates to dry overnight
SECTION: Prepare OP50 seeded NGM plates
6. Seed each NGM plate with 3 ml OP50 liquid culture.
SECTION: Prepare OP50 seeded NGM plates
7. Cover as much agar surface as possible by moving the plate in first a circular pattern, then a figure 8 pattern.
SECTION: Prepare OP50 seeded NGM plates
8. Dry the OP50 seeded plates at room temperature with the lids on for three to four days until there is no more excess liquid
SECTION: Grow mixed stage cultures of cell sorting strain
9. Identify a 60 mm petri plate culture of the sorting strain that has recently exhausted the E. coli lawn
SECTION: Grow mixed stage cultures of cell sorting strain
10. Chunk the plate into 5 equal pieces
SECTION: Grow mixed stage cultures of cell sorting strain
11. Transfer each chunk to a fresh large NGM OP50 plate with the worm covered surface facing down
SECTION: Grow mixed stage cultures of cell sorting strain
12. Place sorting strain cultures in 20°C incubator for 72-96 hours, until the E. coli lawn is exhausted
SECTION: Expand mixed stage cultures of cell sorting strain
13. Harvest the mixed stage worm population from the 5 plates by washing each plate with ~10 ml of M9
SECTION: Expand mixed stage cultures of cell sorting strain
14. Transfer the worm suspension to a 15 ml conical centrifuge tube
SECTION: Expand mixed stage cultures of cell sorting strain
15. Pellet the worms by centrifuging for 1 min at 2,000 rcf
SECTION: Expand mixed stage cultures of cell sorting strain
16. Wash with additional M9 by aspirating the supernatant and resuspending the worms in fresh M9 to a total volume of 15 ml
SECTION: Expand mixed stage cultures of cell sorting strain
17. Repeat the M9 wash until the supernatant is clear
SECTION: Expand mixed stage cultures of cell sorting strain
18. Measure the approximate concentration of worms in suspension such that the optimal density of worms are seeded onto the plate
SECTION: Expand mixed stage cultures of cell sorting strain
18.1. Shake or vortex the tube to ensure the worms are evenly distributed in the suspension
SECTION: Expand mixed stage cultures of cell sorting strain
18.2. Aspirate 2 ul of worm suspension with a p10 pipette. Pipette the worm suspension up and down at least four times before moving on.
SECTION: Expand mixed stage cultures of cell sorting strain
18.3. Dispense the worm suspension on a clean microscope slide
SECTION: Expand mixed stage cultures of cell sorting strain
18.4. With a cell counter, count the number of worms on the slide under a dissection microscope. Dilute the worm suspension if there are too many to count.
SECTION: Expand mixed stage cultures of cell sorting strain
18.5. Determine the concentration and total number of worms

  
  

SECTION: Expand mixed stage cultures of cell sorting strain
19. Seed 20 fresh large NGM OP50 plates with 5,000 worms per plate

NOTE:
Optimal seeding density may need to be independently determined as lab conditions vary. Optimal worm density should allow for worms to consume the bacterial lawn in time to become gravid without inducing a food deprivation stress response. To determine the optimal seeding density, seed a range of worms on several plates and culture until gravid.
SECTION: Expand mixed stage cultures of cell sorting strain
20. Incubate for 72 hours in a 20°C incubator, until there is a large number of gravid adults
SECTION: First embryo synchronization with hypochlorite solution
21. At the beginning for the day, chunk one recently starved 60 mm N2 plate to a fresh large NGM OP50 plate. This is key step, and will serve as the negative GFP control for cell sorting.
SECTION: First embryo synchronization with hypochlorite solution
22. Harvest mixed stage sorting strain worms from all 20 plates by washing each plate individually with ~10 ml of M9
SECTION: First embryo synchronization with hypochlorite solution
22.1. Transfer mixed stage worm suspension to a 15 ml conical centrifuge tube
SECTION: First embryo synchronization with hypochlorite solution
22.2. Pellet the worms by centrifuging for 1 minute at 2,000 rcf
SECTION: First embryo synchronization with hypochlorite solution
22.3. Aspirate the supernatant
SECTION: First embryo synchronization with hypochlorite solution
22.4. Harvest worms from another plate as outline above
SECTION: First embryo synchronization with hypochlorite solution
22.5. Resuspend the worm pellet with worm suspension from the newly washed plate
SECTION: First embryo synchronization with hypochlorite solution
22.6. Repeat this process until worms have been harvested from all 20 plates into a single 15ml tube
SECTION: First embryo synchronization with hypochlorite solution
23. Once all plates have been harvested, continue washing the worm pellet with fresh M9 to remove excess E. coli by pelleting and resuspending in fresh M9. The final worm pellet yield should be 1 to 2 ml.
SECTION: First embryo synchronization with hypochlorite solution
24. Once the worm suspension is free of E. coli, centrifuge again and remove all M9 supernatant from the worm pellet
SECTION: First embryo synchronization with hypochlorite solution
25. Resuspend the worm pellet in 8 ml of H2O
SECTION: First embryo synchronization with hypochlorite solution
26. Add 0.9 ml of Sodium Hypochlorite Solution (Ricca Chemical, 7495.7-32) and 1.44 ml of 5N NaOH to the worm suspension
SECTION: First embryo synchronization with hypochlorite solution
27. Resuspend the worm pellet with brief vigorous vortexing 
SECTION: First embryo synchronization with hypochlorite solution
28. Incubate at room temperature for 6 to 8 minutes. While incubating shake the tube or place on a nutator. The time to bleach the worms depends on the worm pellet volume, with larger worm pellets taking longer. Do not incubate for longer than 8 minutes. 
SECTION: First embryo synchronization with hypochlorite solution
29. Monitor the progression of the hypochlorite treatment. Larval worms should dissolve, adult worms will begin to break at the vulva and release embryos. I typically monitor the treatment by looking through the tube under a dissection microscope. Aliquots of the worm suspension can also be taken throughout the process and viewed on a microscope slide.
SECTION: First embryo synchronization with hypochlorite solution
30. Once the worms are sufficiently dissolved, centrifuge the tube for 30 seconds at 2,000 rcf
SECTION: First embryo synchronization with hypochlorite solution
31. Decant the supernatant and wash the embryo pellet by adding 15 ml of M9 to quench the hypochlorite treatment
SECTION: First embryo synchronization with hypochlorite solution
32. Wash the bleached embryos a second time. Centrifuge the tube for 30 seconds at 2,000 rcf to pellet the embryo suspension. Decant the supernatant and resuspend the embryo pellet in 15 ml of M9.
SECTION: First embryo synchronization with hypochlorite solution
33. Wash the bleached embryos a third time. Centrifuge the tube for 30 seconds at 2,000 rcf to pellet the embryo suspension. Decant the supernatant and resuspend the embryo pellet in 15 ml of M9.
SECTION: First embryo synchronization with hypochlorite solution
34. Wash the bleached embryos a fourth time. Centrifuge the tube for 30 seconds at 2,000 rcf to pellet the embryo suspension. Decant the supernatant and resuspend the embryo pellet in 15 ml of M9.
SECTION: First embryo synchronization with hypochlorite solution
35. Measure the approximate concentration of embryos in suspension
SECTION: First embryo synchronization with hypochlorite solution
35.1. Shake or vortex the tube to ensure the embryos are evenly distributed in the suspension
SECTION: First embryo synchronization with hypochlorite solution
35.2. Aspirate 2 ul of embryo suspension with a p10 pipette. Pipette the embryo suspension up and down at least four times before moving on.
SECTION: First embryo synchronization with hypochlorite solution
35.3. Dispense the embryo suspension on a clean microscope slide
SECTION: First embryo synchronization with hypochlorite solution
35.4. With a cell counter, count the number of embryos on the slide under a dissection microscope. Dilute the embryo suspension if there are too many to count. See step 18.5 for the formula to determine the embryo concentration.
SECTION: First embryo synchronization with hypochlorite solution
36. Seed 20 large NGM/OP50 plates with 5,000 embryos. Incubate at 20°C for approximately 72 hours until worms are gravid.
SECTION: Second embryo synchronization with hypochlorite solution
37. Harvest both synchronized sorting strain worms and mixed stage N2 worms in parallel by washing individual plates with ~10 ml of M9 and collecting in two separate 15 ml tubes
SECTION: Second embryo synchronization with hypochlorite solution
38. Transfer the worm suspension to a 15 ml conical centrifuge tube
SECTION: Second embryo synchronization with hypochlorite solution
39. Pellet the worms by centrifuging for 1 minute at 2,000 rcf
SECTION: Second embryo synchronization with hypochlorite solution
40. Discard the supernatant
SECTION: Second embryo synchronization with hypochlorite solution
41. Resuspend the worm pellet with worm suspension from another large plate
SECTION: Second embryo synchronization with hypochlorite solution
42. Repeat this process until worms have been harvested from all 20 plates for the sorting strain and 1 N2 plate
SECTION: Second embryo synchronization with hypochlorite solution
43. Once all plates have been harvested, continue washing the worm pellet with fresh M9 to remove excess E. coli. The final worm pellet yield for the sorting strain should be 1 to 2 ml.
SECTION: Second embryo synchronization with hypochlorite solution
44. Once the worm suspension is free of E. coli, centrifuge again and remove all M9 supernatant from the worm pellet
SECTION: Second embryo synchronization with hypochlorite solution
45. Resuspend the worm pellet in 8 ml of H2O
SECTION: Second embryo synchronization with hypochlorite solution
46. Add 0.9 ml of Sodium Hypochlorite Solution (Ricca Chemical, 7495.7-32) and 1.44 ml of 5N NaOH to the worm suspension
SECTION: Second embryo synchronization with hypochlorite solution
47. Resuspend the worm pellet with brief vigorous vortexing 
SECTION: Second embryo synchronization with hypochlorite solution
48. Incubate at room temperature for 6 to 8 minutes. While incubating shake the tube or place on a nutator. The time to bleach the worms depends on the worm pellet volume, with larger worm pellets taking longer. Do not incubate for longer than 8 minutes. 
SECTION: Second embryo synchronization with hypochlorite solution
49. Monitor the progression of the hypochlorite treatment. Larval worms should dissolve, adult worms will begin to break at the vulva and release embryos. I typically monitor the treatment by looking through the tube under a dissection microscope. Aliquots of the worm suspension can also be taken throughout the process and viewed on a microscope slide.
SECTION: Second embryo synchronization with hypochlorite solution
50. Once the worms are sufficiently dissolved, centrifuge the tube for 30 seconds at 2,000 rcf
SECTION: Second embryo synchronization with hypochlorite solution
51. Decant the supernatant and resuspend the embryo pellet in 15 ml of M9 to quench the hypochlorite treatment
SECTION: Second embryo synchronization with hypochlorite solution
52. Wash the bleached embryos a second time. Centrifuge the tube for 30 seconds at 2,000 rcf to pellet the embryo suspension. Decant the supernatant and resuspend the embryo pellet in 15 ml of M9.
SECTION: Second embryo synchronization with hypochlorite solution
53. Wash the bleached embryos a third time. Centrifuge the tube for 30 seconds at 2,000 rcf to pellet the embryo suspension. Decant the supernatant and resuspend the embryo pellet in 15 ml of M9.
SECTION: Second embryo synchronization with hypochlorite solution
54. Wash the bleached embryos a fourth time. Centrifuge the tube for 30 seconds at 2,000 rcf to pellet the embryo suspension. Decant the supernatant and resuspend the embryo pellet in 15 ml of M9.
SECTION: Second embryo synchronization with hypochlorite solution
55. The final embryo yield should be approximately 0.01 ml for the wildtype N2 stain and 0.2ml for the fluorescent sorting strain.
SECTION: Second embryo synchronization with hypochlorite solution
56. For embryo stage FACS experiments, move on to the embryo dissociation protocol 
SECTION: Second embryo synchronization with hypochlorite solution
57. For post-embryonic stage FACS experiments, move on to Step 58 for L1 stage experiments or Step 65 for L3 stage experiments
SECTION: Synchronized L1 culture
58. Incubate the synchronized embryos in 15 ml M9 overnight rotating in 20°C incubator for 24 hours
SECTION: Synchronized L1 culture
59. Feed the synchronized L1 sorting strain worms six hours before beginning the L1 dissociation protocol

Note:
This step is necessary to reduce any observable starvation-induced responses in the measured transcriptional data. Negative control N2 worms can remain incubating in M9 suspension.
SECTION: Synchronized L1 culture
60. Pellet the L1 sorting strain worms and resuspend in 500 ul of M9. Transfer equal volumes of the synchronized L1 sorting strain worms onto two large peptone enriched NGM OP50 plates
SECTION: Synchronized L1 culture
61. Feed the synchronized L1 worms for 6 hours in a 20°C incubator.
SECTION: Synchronized L1 culture
62. Harvest the synchronized L1 worms by washing the plates with fresh M9. Pellet the worms for 1 min at 2,000 rcf. Discard the supernatant and repeat M9 washes until the supernatant is free of visible E. coli.
SECTION: Synchronized L1 culture
63. For both fluorescent sorting strain and wildtype strain, pass the harvested L1 suspension through a 20 micron filter. This will filter any contaminating debris (agar chunks, partially bleached worm chunks) and any unhatched or dead embryos.
SECTION: Synchronized L1 culture
64. Move on to the L1 stage dissociation protocol


SECTION: Synchronized L3 culture
65. Synchronize the embryos to the L1 stage by incubating the embryos for 24 hours suspended in M9, rotating in a 20°C incubator
SECTION: Synchronized L3 culture
66. Measure the approximate concentration of L1 worms in suspension
SECTION: Synchronized L3 culture
66.1. Shake or vortex the tube to ensure the worms are evenly distributed in the suspension
SECTION: Synchronized L3 culture
66.2. Aspirate 2 ul of worm suspension with a p10 pipette. Pipette the worm suspension up and down at least four times before moving on.
SECTION: Synchronized L3 culture
66.3. Dispense the worm suspension on a clean microscope slide
SECTION: Synchronized L3 culture
66.4. With a cell counter, count the number of worms on the slide under a dissection microscope. Dilute the embryo suspension if there are too many to count.
SECTION: Synchronized L3 culture
67. Seed the plates with a worm suspension volume between 100 and 500 ul. Concentrate or dilute the worm suspension accordingly. To avoid generating stressed clumps of developmentally delayed worms, spot the worm suspension evenly across the OP50 lawn.
SECTION: Synchronized L3 culture
67.1. For the sorting strain worms, seed at least 10 large peptone enriched NGM OP50 plates with 30,000 L1 worms.
SECTION: Synchronized L3 culture
67.2. For the wildtype worms, seed at least 5 large peptone enriched NGM OP50 plates with 30,000 L1 worms
SECTION: Synchronized L3 culture
68. Incubate the worms for 48 hours in a 20C incubator until worms visibly reach the L3 stage.
SECTION: Synchronized L3 culture
69. Move on to the L3 stage dissociation protocol