C. elegans Synchronizer SystemProtocol for synchronization of L1 nematodes

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Warning:

! Make sure to protect the filters when not in use by placing a 90mm petri dish cap om each side. Also when working with for example a pipette, be careful not to puncture the filters. Before use, please read the general instructions and remarks paragraph.

Preparation:

a. Sterilize the filters, funnel, crystallizing dish, etc, with 70% ethanol and rinse once with sterile M9/S-media. b. For optimal yield and synchronization the majority of the C. elegans culture should consists of gravid adults and eggs. c. See also section ‘ General Instructions and Remarks’.

Protocol:

A: Stabilizing1. Wash the worms off plates or use a liquid culture of worms.2. Wash the worms once by sedimentation.3. Place the stabilization filter in the glass funnel with the filter facing downwards. Make sure the silicone tube is placed in a

‘waste’ container.4. Pour the worms onto the stabilization filter.5. Wash the worms in the stabilization filter, multiple times with sterile M9/S-media (3-4 times).6. After the washing steps have been completed, close/pinch the silicone waste tube using a Hemostat Locking Clamp or

similar device.7. Fill the stabilization filter and funnel with M9/S-media to a level just a few millimeters above the filter surface.8. Let the worms sediment for 5 minutes.9. Release the Hemostat Locking Clamp and discard the content of the waste container.10. If using a Liquid culture continue to ‘B1: Harvest from Liquid culture’.11. If using a NGM culture continue to ‘B2: Harvest from NGM culture’.

B1: Harvest from Liquid culture12. Place the Harvest filter in the crystallizing dish with spout.13. Transfer the worms and eggs from the Stabilizing filter in to the Harvest filter:

This can be done with a pipet from the surface of the Stabilizing filter or by removing the Stabilizing filter from the funnel and turn it upside down holding it above the Harvest filter and with a gentle wash from the back side of the filter using M9/S-media (aprox 5ml), flush the worms and eggs out of the Stabilizing filter and in to the Harvest filter.

14. Carefully lift the Harvest filter a few millimeters out of the crystallizing dish and add a small layer of liquid (M9/S-media) on the bottom of the dish and on top of the filter. The entire bottom and top of the filter should be covered to let the L1 nematodes through.

15. Close off the top of the Harvest filter with a Petri dish.16. Leave the Harvest filter for at least 5-10 minutes or a maximum of 12 hours depending on the amount of nematodes and

level of synchronization required.17. When harvest is completed take the Harvest filter out of the Crystallizing dish and store the filter safely in a Petri dish (filter

facing downwards in the dish). 18. The synchronized L1 can now be collected from the Harvest Crystallizing dish.19. If needed clean the synchronized L1’s using the Cleaning buffer, transfer the L1’s to a 50 mL tube and add 10 mL Cleaning

buffer, incubate for 5 minutes. 20. Wash twice with 10 mL M9/S-media through table-top centrifugation (1200rpm for 3 min)21. Re-suspend the washed pellet in the desired volume of M9/S-media buffer.

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B2: Harvest from NGM culture (1)

12. Pipet the liquid and worms from the stabilization filter into (multiple) 15ml tubes.13. Keep the tubes rotation overnight while the worms lay eggs and the L1’s hatch .14. Place the Harvest filter in the crystallizing dish with spout.15. Pipet the supernatant with L1's and Eggs onto the Harvest filter.16. Carefully lift the Harvest filter a few millimeters out of the crystallizing dish and add a small layer of liquid (M9/S-media) on

the bottom of the dish and on top of the filter. The entire bottom and top of the filter should be covered to let the L1 nematodes through.

17. Close off the top of the Harvest filter with a Petri dish.18. Leave the Harvest filter for at least 5-10 minutes or a maximum of 12 hours depending on the amount of nematodes and

level of synchronization required.19. When harvest is completed take the Harvest filter out of the Crystallizing dish and store the filter safely in a Petri dish (filter

facing downwards in the dish). 20. The synchronized L1 can now be collected from the Harvest Crystallizing dish.21. If needed clean the synchronized L1’s using the Cleaning buffer, transfer the L1’s to a 50 mL tube and add 10 mL Cleaning

buffer, incubate for 5 minutes. 22. Wash twice with 10 mL M9/S-media through table-top centrifugation (1200rpm for 3 min)23. Re-suspend the washed pellet in the desired volume of M9/S-media buffer.

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General instructions and remarks:

Sterilizing the filters and funnel:

Sterilize the filters by washing them with 70% ethanol and rinse once with sterile M9/S-media. Sterilize the funnel by washing it with 70% ethanol and optional rinse once with sterile M9/S-media. Work in a flow hood or with a flame in order to prevent contamination.

Cleaning / descaling the filters:

Only use demi water. NEVER use standard tap water as it will scale the filters. If the filters become clogged the following cleaning procedure is recommended:

a) in order to remove organic materials a few minutes in a 1 M NaOH solution will typically suffice.b) if there is ‘scaling’ NEVER use an acid solution as it will have the potential of damaging the filters. Instead use a small 40khz ultrasonic cleaning device with a ‘general purpose’ cleaning solution that has pH of 7 or higher. If not sure just use demi-water with a drop of general purpose detergent and gently move / tilting the filter for 1 to 2 minutes in the ultrasonic cleaning device. To verify the result check the filter before and after under a microscope.

Media and Buffer preparation:

Harvest buffer 50 mL: Basal, S media or water (see Wormbook for preparation)

Cleaning buffer 50 mL: M9 + Streptomycin + Nystatin –solution

- Streptomycin stock 100 mg/mL add 1 gram of Strep to 10 mL of dH2O, filter sterilize the solution and store at 4˚C

- Nystatin suspension 10 mg/mLfor 50 mL, add 0.5 gram Nystatin in 70% ethanol in dH2O (Shake before use) store at 4˚C

- Prepare 50 mL of sterile M9 bufferadd 50 µL Streptomycin stock 100 mg/mL and 50 µL Nystatin suspension 10 mg/mL

Protocol revisions and updates:

The latest protocols and documentation can be downloaded at https://www.nemasync.com/documentation

Credits:

(1) The protocol for NGM Harvest has been optimized by Ms. Renée Seinstra – Research Analyst UMCG - European Institute for the Biology of Aging (ERIBA) as this will increase the amount of egg’s overnight when using NGM plates as the breeding source.

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