Sep 23, 2022
Version 2
Synchronized C. elegans culture on NGM plates for FACS isolation of intestine cells V.2
- 1Colorado State University, Fort Collins
Protocol Citation: Robert TP Williams, Erin Osborne Nishimura 2022. Synchronized C. elegans culture on NGM plates for FACS isolation of intestine cells. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv59zjng1b/v2Version created by Robert TP Williams
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: September 22, 2022
Last Modified: September 23, 2022
Protocol Integer ID: 70391
Keywords: C. elegans, intestine, FACS, single cell suspension, cell dissociation
Funders Acknowledgement:
National Institutes of Health
Grant ID: R35GM124877
Bridge to Doctorate at Colorado State University
Grant ID: 1612513
National Science Foundation
Grant ID: 2143849
Webb-Waring Biomedical Research Award
Grant ID: E.O.N.
Abstract
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.
Materials
Strains:
- OP50 E. coli
- FACS control C. elegans strain, i.e. N2
- FACS sorting C. elegans strain, i.e. JM149 caIs71[elt-2p::GFP::HIS-2B::unc-54 3'UTR + rol-6(su1006)]
Reagents:
- LB Broth Mix (Genesee 11-120)
- M9 buffer
- 3 g KH2PO4 (Sigma-Aldrich P0662)
- 6 g Na2HPO4 (Thermo Fisher S373)
- 5 g NaCl (Sigma-Aldrich S9888)
- 1 ml 1 M MgSO4 (Sigma-Aldrich 208094)
- H2O to 1 liter
- NGM plates
- (complete protocol: http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html#d0e214)
- 3g NaCl (Sigma-Aldrich S9888)
- 17g agar (Genesee 20-249)
- 2.5g peptone (VWR 89406-350)
- 975g H2O (sterile and deionized)
- 1ml 1M CaCl2 (Sigma-Aldrich C3306)
- 1ml 5mg/ml cholesterol (Fisher 501848291)
- 1ml 1M MgSO4 (Sigma-Aldrich 208094)
- 25ml 1M KPO4 Buffer pH 6.0 (108.3 g KH2PO4, 35.6 g K2HPO4, H2O to 1 litre) (Sigma-Aldrich P0662, P3786
- Peptone enriched NGM: in recipe above use 20g peptone instead of 2.5g peptone
- Bleaching solution
- Sodium Hypochlorite Solution, 6% available chlorine (Ricca Chemical, 7495.7-32)
- 5N NaOH (Fisher S318-100)
Consumables:
- 150 mm petri dishes "large plates" (Corning 351058)
- 15 ml centrifuge tubes (Peak PS-695)
Equipment:
- Swinging bucket rotor refrigerated centrifuge (Eppendorf 5810R)
- Pipet-Aid (VWR 89166-464)
- 20°C incubator (Caron 7001-28-1)
Prepare OP50 seeded NGM plates
Prepare OP50 seeded NGM plates
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
Grow mixed stage cultures of cell sorting strain
Grow mixed stage cultures of cell sorting strain
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
4d
Expand mixed stage cultures of cell sorting strain
Expand mixed stage cultures of cell sorting strain
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
First embryo synchronization with hypochlorite solution
First embryo synchronization with hypochlorite solution
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
1h
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.
Second embryo synchronization with hypochlorite solution
Second embryo synchronization with hypochlorite solution
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 post-embryonic stage FACS experiments, move on to Step 58 for L1 stage experiments or Step 65 for L3 stage experiments
Synchronized L1 culture
Synchronized L1 culture
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.
Synchronized L3 culture
Synchronized L3 culture
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