Immobilize and Recover live C. elegans without chemical anesthetics

NemaGel is an easy way to completely immobilize C. elegans for imaging purposes with the ability to reverse the immobilization and recover the worms.

  • No chemical anesthetics
  • Rapid immobilization (< 5 minutes) upon temperature shift
  • Reversible with nearly 100% recovery of worms 
  • Suitable for low and high magnification (10x-100x)

Comparison of Conventional Gel vs. NemaGel

N2 worms were acquired on the NemaImager with a 10x objective at 25°C. Adult worms are fully immobilized
using NemaGel (right) vs. animal still moving with a standard gel (left).

Advantages

NemaGel is a temperature-dependent (temperature is adjusted to 25°C) and reversible immobilization solution for imaging live C. elegans, immobilization occurs immediately upon temperature adjustment with NemaGel.

Unlike other common C. elegans immobilization solution, such as levamisole or sodium azide, which may impact viability and morphology, particularly when trying to recover worms, NemaGel is chemical anesthetics free.  

Recovering worms anesthetics-free and glue-free allows for repeated imaging of the same worm over a time span of several days, propagation of specific worms post-imaging, or imaging identical worms pre-post drug treatment.

Our fluorescent staining kits allow you to stain your worms for fluorescent imaging.

Image captured on the NemaImager with a 20x objective at 25°C. Animals express fluorescent proteins from integrated arrays with the GCamp6 under the myo-2 promoter in the body wall muscles and the tdTomato under pm6 in the pharynx.

C.elegans neurons

Worms were immobilized using NemaGel at 25°C, enabling the image to be captured on the NemaImager at 100x magnification. Neurons of N2 worms stained with WormDye Neuro Red – DiI.

How Does It Work?

NemaGel uses polystyrene microbeads (30μm) to allow for a uniform pressure on the coverslip without damaging the C. elegans.  The uniform pressure allows for more complete compression and immobilization of the worm sample in the gel.

NemaGel will form into a gel above 10°C . It will re-liquify when cooled to below 10°C. The gel should remain stable during autoclaving. NemaGel is provided in an easy to use dropper bottle with simple protocols.

Ordering Information

DescriptionQtyPackagingPriceCat. No.
NemaGel - 40 uses/20ml 1 2 vials $60.00NGEL-200
NemaGel - 80 uses/40ml14 vials$110.00NGEL-400
NemaGel - 100 uses/50ml15 vials$130.00NGEL-500
NemaGel - 120 uses/60ml16 vials$150.00NGEL-600

Shipping Details:
- The product will be shipped at room temperature.
US: $15 flat rate via USPS or use your own shipping account.
Canada: $35 or use your own shipping account.
- Other Countries: $120 or use your own shipping account.

Frequently Asked Questions (FAQs)

NemaGel is highly compatible with fluorescent microscopy. NemaGel is perfectly transparent. Additionally, due to the slight compression of worms, a noticeably larger portion (up to 30%) of the worm muscle cells are located within the imaging focal plane. The effect of this is an improvement in visualization of the internal, allowing for a more extensive visualization of both cellular subsystems and organs including the mitochondrial network and gonads.

Conventional immobilization methods rely on anesthetic drugs. Examples for paralysis include; sodium azide, phenoxypropanol, and tetramisole. Fixation with glue has also been indicated. These methods often dramatically disrupt normal physiological activities of the worms (a good example is sodium azide disruption of cellular activity). Both alternative immobilization methods result in inhibited natural worm development. Even if the worms can be recovered, they are no longer fully viable, or usable after imaging.

C. elegans larvae (L1 to L4) are effectively immobilized (and recoverable) in the NemaGel even though their diameter is smaller than the diameter of the microbeads.

To effectively image young adult to old C. elegans, compression is required to effectively immobilize them. The NemaGel allows the C. elegans worm to be effectively compressed to a level that is not harmful.

Official results from testing applications for zebrafish will be available in the coming weeks. Overlaying the cold NemaGel over the embryo should not impact the health of the zebrafish (according to this citation the zebrafish embryo can be chilled to 4C for hours before measurable impact  - https://www.ncbi.nlm.nih.gov/pubmed/19406462).

It should also be noted that use of NemaGel with an anesthetic requires mixing the chemical with NemaGel while it is still cold, prior to laying it over the zebrafish embryo.

It can be used with drosophila larvae. This has been successfully performed by outside scientists. We have not replicated this ourselves internally. Even though Drosophila larvae are significantly larger than C. elegans (and, theoretically stronger), they are also subjected to higher viscous forces. There is also a major difference in the natural body motion pattern. In the case of C. elegans worms this is composed of lateral undulations. Whereas D. melanogaster larvae locomotion is induced by circular muscles squeezing, and extending the larval body along its axis. When measuring the frequency of small lateral head thrashes researchers found that D. melanogaster larvae can be sufficiently immobilized.

The NemaGel has been extensively tested for use with C. elegans. However, it has the potential to be used for immobilization and recovery of a wide range of microorganisms, such as Drosophila and Trypanosoma brucei.

NemaGel is a biocompatible, triblock copolymer mixed with polystyrene microbeads. In solution, it undergoes thermogelling at a specific gelation temperature that is below room temperature. The polystyrene microbeads are incorporated to allow uniform pressure on the coverslip without damaging the worm. Between the gelling of the viscous copolymer and the uniform mild compression the worms are physically immobilized. In our observations this has had minimal impact on the internal processes during a reasonable observation period (20 minutes). If you zoom in on the video above, you can still see internal activity. 

After NemaGel immobilization for 2 hours we did not observe a significant change in fertility post-recovery with respect to the control group, which is an indication of no obvious adverse physiological effects.

It just depends how much immobilization you need. To achieve complete immobilization you would need to use the coverslip. You will experience some reduction in movement without it.

The technique allows recovery of the worms which is a significant advantage over conventional anesthetics- or glue-based methods. This can be useful for repeated imaging of the same worm over a time span of several days, propagation of specific worms post-imaging, or imaging identical worms pre-post drug treatment.

NemaGel is a biocompatible, triblock copolymer. The gel is liquid when cold and solid at room temperature. When a cold drop is added onto the worms, it quickly becomes solid and freezes them. The microbeads prevent squeeze the worms in place but prevent them from getting too squished. To recover the worms, the solution can be diluted away until the worms are free again. Additionally, polystyrene microbeads are incorporated to allow uniform pressure on the coverslip without damaging the worm. Uniform pressure also allows for a more complete immobilization of the worm within the NemaGel.

You will need to work quickly. NemaGel will solidify when added rapidly (<2 minutes). During recovery they will become free equally quickly (<2min).

We have not tested the salt impact in our labs.  However, salt or salt-water will alter the composition of the NemaGel, such that its gelation properties will change.  The temperature at which the NemaGel transitions from a liquid to a gel (and vice versa) will alter, as will the internal environment of the gel; it is possible that addition of salt may also make the gel somewhat hygroscopic. 

24 months. We recommend using within a year after opening.

 

NemaGel is stable at room temperature. However, our product is shipped on an ice pack to prevent extreme temperature fluctuations. For long term storage, it is best to store at 4°C upon arrival. Avoid freezing. Allow 1-4 hours for the NemaGel to become completely liquid prior to your first use.

Protocols

  1. Add 10ul of M9 to the center of a slide.
  2. Pick C. elegans to the M9 drop (Alternative, use a suspended solution of worms)
  3. Surround M9 with cooled (4°C) NemaGel solution. Click to view.
  4. Add 1 drop of NemaGel to the center of a coverslip, then apply to the center of the slide.
  5. Heat the slide to 25°C and image.
  • Remove the coverslip from the slide.  Rinse both the coverslip and slide with 30ul M9 onto an agar plate. 
  • C. elegans will slowly recover movement (fully mobile in 4 minutes).
  • Work quickly when applying the NemaGel as it solidifies rapidly (~30s) at room temperature.
  • You can use a bead water bath or temperature block to obtain 25°C immobilization.
  • For some imaging purposes, room temperature may provide sufficient immobilization.

Looking for a compact-sized microscope? Check out the Etaluma LS560 Microscope.