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Agar pad method for C. elegans temperature-sensitive embryos

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

1/ Early-stage embryo dissection

Pick young adults into pre-cooled M9 medium (16°C).

Dissect them by cutting at both end of the uterus using a scalpel and #15 blade to release the embryos.

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

2/ Mounting on an agarose pad

Add a small drop of 2% agarose on a 170µm thick cover slip and cover it with a perpendiculary placed slide (not a cover slip!) to make the agarose pad.

Once cooled, twist slides relative to each other to expose the pad. It must remain on the cover slip.

3/ Two possibilities next to fit with our CherryTemp control unit

Add embryos in a 5µl drop of M9, gently add the agarose pad on top. Top with our emperature control chip. Gently clamp the system using our insert.

OR

Directly add embryos, top with a cover slip, turn the slides upside down. Place the “slide sandwich” in our insert. Add a drop of immersion oil and top with our temperature control chip. Gently clamp the system using our insert.

methods-c-elegans-embryo-temperature-control-agar-pad-3

Schematics of embryos on an agarose pad

 Methods adapted from J. Dumont and J.C. Canman:

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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