Synchronized C. elegans culture on NGM plates for FACS isolation of intestine cells

Using sterile technique, pick an OP50 colony and inoculate a 250 ml bottle of sterile LB.

Incubate OP50 liquid culture at 37°C overnight

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.

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.

Pour molten NGM into large plates. Each liter should make 20 plates, for a total of 60 large plates

Allow plates to dry overnight

Seed each NGM plate with 3 ml OP50 liquid culture.

Cover as much agar surface as possible by moving the plate in first a circular pattern, then a figure 8 pattern.

Dry the OP50 seeded plates at room temperature with the lids on for three to four days until there is no more excess liquid

Identify a 60 mm petri plate culture of the sorting strain that has recently exhausted the E. coli lawn

Chunk the plate into 5 equal pieces

Transfer each chunk to a fresh large NGM OP50 plate with the worm covered surface facing down

Place sorting strain cultures in 20°C incubator for 72-96 hours, until the E. coli lawn is exhausted

Harvest the mixed stage worm population from the 5 plates by washing each plate with ~10 ml of M9

Transfer the worm suspension to a 15 ml conical centrifuge tube

Pellet the worms by centrifuging for 1 min at 2,000 rcf

Wash with additional M9 by aspirating the supernatant and resuspending the worms in fresh M9 to a total volume of 15 ml

Repeat the M9 wash until the supernatant is clear

Measure the approximate concentration of worms in suspension such that the optimal density of worms are seeded onto the plate

Shake or vortex the tube to ensure the worms are evenly distributed in the suspension

Aspirate 2 ul of worm suspension with a p10 pipette. Pipette the worm suspension up and down at least four times before moving on.

Dispense the worm suspension on a clean microscope slide

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.

Determine the concentration and total number of worms

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.

Incubate for 72 hours in a 20°C incubator, until there is a large number of gravid adults

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.

Harvest mixed stage sorting strain worms from all 20 plates by washing each plate individually with ~10 ml of M9

Transfer mixed stage worm suspension to a 15 ml conical centrifuge tube

Pellet the worms by centrifuging for 1 minute at 2,000 rcf

Aspirate the supernatant

Harvest worms from another plate as outline above

Resuspend the worm pellet with worm suspension from the newly washed plate

Repeat this process until worms have been harvested from all 20 plates into a single 15ml tube

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.

Once the worm suspension is free of E. coli, centrifuge again and remove all M9 supernatant from the worm pellet

Resuspend the worm pellet in 8 ml of H2O

Add 0.9 ml of Sodium Hypochlorite Solution (Ricca Chemical, 7495.7-32) and 1.44 ml of 5N NaOH to the worm suspension

Resuspend the worm pellet with brief vigorous vortexing

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.

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.

Once the worms are sufficiently dissolved, centrifuge the tube for 30 seconds at 2,000 rcf

Decant the supernatant and wash the embryo pellet by adding 15 ml of M9 to quench the hypochlorite treatment

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.

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.

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.

Measure the approximate concentration of embryos in suspension

Shake or vortex the tube to ensure the embryos are evenly distributed in the suspension

Aspirate 2 ul of embryo suspension with a p10 pipette. Pipette the embryo suspension up and down at least four times before moving on.

Dispense the embryo suspension on a clean microscope slide

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.

Seed 20 large NGM/OP50 plates with 5,000 embryos. Incubate at 20°C for approximately 72 hours until worms are gravid.

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

Transfer the worm suspension to a 15 ml conical centrifuge tube

Pellet the worms by centrifuging for 1 minute at 2,000 rcf

Discard the supernatant

Resuspend the worm pellet with worm suspension from another large plate

Repeat this process until worms have been harvested from all 20 plates for the sorting strain and 1 N2 plate

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.

Once the worm suspension is free of E. coli, centrifuge again and remove all M9 supernatant from the worm pellet

Resuspend the worm pellet in 8 ml of H2O

Add 0.9 ml of Sodium Hypochlorite Solution (Ricca Chemical, 7495.7-32) and 1.44 ml of 5N NaOH to the worm suspension

Resuspend the worm pellet with brief vigorous vortexing

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.

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.

Once the worms are sufficiently dissolved, centrifuge the tube for 30 seconds at 2,000 rcf

Decant the supernatant and resuspend the embryo pellet in 15 ml of M9 to quench the hypochlorite treatment

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.

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.

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.

The final embryo yield should be approximately 0.01 ml for the wildtype N2 stain and 0.2ml for the fluorescent sorting strain.

For embryo stage FACS experiments, move on to the embryo dissociation protocol

For post-embryonic stage FACS experiments, move on to Step 58 for L1 stage experiments or Step 65 for L3 stage experiments

Incubate the synchronized embryos in 15 ml M9 overnight rotating in 20°C incubator for 24 hours

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.

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

Feed the synchronized L1 worms for 6 hours in a 20°C incubator.

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.

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.

Move on to the L1 stage dissociation protocol

Synchronize the embryos to the L1 stage by incubating the embryos for 24 hours suspended in M9, rotating in a 20°C incubator

Measure the approximate concentration of L1 worms in suspension

Shake or vortex the tube to ensure the worms are evenly distributed in the suspension

Aspirate 2 ul of worm suspension with a p10 pipette. Pipette the worm suspension up and down at least four times before moving on.

Dispense the worm suspension on a clean microscope slide

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.

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.

For the sorting strain worms, seed at least 10 large peptone enriched NGM OP50 plates with 30,000 L1 worms.

For the wildtype worms, seed at least 5 large peptone enriched NGM OP50 plates with 30,000 L1 worms

Incubate the worms for 48 hours in a 20C incubator until worms visibly reach the L3 stage.

Move on to the L3 stage dissociation protocol