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Agarose pad method for C. elegans temperature-sensitive embryos

Agarose pad method for controlling C. elegans temperature-sensitive embryos

1/ Prepare your thin agar pad

To get the full efficiency of our CherryTemp temperature controller and ensure the accurate control we provide, samples (cells/embryos) must be placed close to the thermalization chip (<500µm).

Following this protocole, you will prepare thin agar pads perfectly suitable with our CherryTemp chip holders and inserts.

  1. Place 3 slides next to each others and stick standard lab tape lengthwize on the two external slides (Fig. 1).
  2. Drop about 50-100µl of melted agarose on the middle slide (Fig. 2).
  3. Cover on top with a clean slide, perpendicular to the 3 others (Fig. 3); apply gentle pressure (Fig. 4).


Figure 1: Align 3 slides and stick lab tape (red) on the 2 external ones.

Figure 2: Drop melted agarose on the middle one.

Figure 3: Top the agarose with another clean slide, perpendicularly.

Figure 4: Apply gentle pressure and allow agarose to solidify.

2/ Early-stage embryo dissection
  1. Pick young adults into pre-cooled M9 medium (16°C).
  2. Dissect them by cutting at both end of the uterus using a scalpel and #15 blade to release the embryos.
  3. Choose the embryos of the desired stage and transfer them to a cover slip using a mouth pipette.
Early-stage C. elegans embryos dissection for live-imaging analysis

Early-stage C. elegans embryos dissection for live-imaging analysis

3/ Mounting cells on an agarose pad
  1. Once the agarose is cooled and when ready to image, twist slides relative to each other to expose the pad (Fig. 5).
  2. Place the CherryTemp chip upside down (engravements down, cover slip and liquid microfluidic up) and transfer the pad on it.
  3. Add embryos in a 5µl drop of M9.
  4. Top with a 170µm cover slip (or a 140µm for SR-microscopy.
  5. Gently clamp the system into our chip holder and go to your upright microscope
  6. Turn the system upside-down, place it in the provided insert and gently clamp.

Figure 5: Gently remove the top slide.

Figure 6: Transfer the pad onto our chip. It must be centered on the thermalizing pattern.

Figure 7: Drop the embryos on the pad.

Figure 8: Top with a high-resolution cover slip.

4/ Place the mounting system on either an upright or an inverted configuration
  1. Gently clamp the system into our chip holder and go to your upright microscope (Fig. 9)
  2. Turn the system upside-down, place it in the provided insert, gently clamp and instal on your inverted stage (Fig. 10)

Figure 9: Use a dedicated chip holder.
(from bottom to top: lower holder part, chip manifoldthermalizing patternclosing cover slipagarose padcells, top cover slip, top holder parts)

Upright configuration2

CherryTemp mounting system in an UPRIGHT configuration

Figure 10: Use customized inserts to maintain the system.

(from bottom to top: chip insert, cover slip, embryos, agarose pad, closing cover slip, thermalizing patternchip manifold)

Inverted configuration2

CherryTemp mounting system in an INVERTED configuration

Methods adapted from J. Dumont and J.C. Canman labs (2016) as well as various protocoles available on the Hyman lab and the worm atlas website and from our user’s feedback.

T. Davies, S. Sundaramoorthy, S.N. Jordan,M. Shirasu-Hiza, J. Dumont and J.C. Canman, 2016, “Using fast-acting temperature-sensitive mutants to study cell division in Caenorhabditis elegans“, Methods in Cell Biology, Volume 137, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2016.05.004 


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