C. elegans Blastomere Dissection: A Method to Remove the Eggshell and Dissociate Embryonic Cells

– For this dissection, use two micropipettes: one with an opening approximately the same width as an egg and one that is twice as wide, and a series of solutions organized in wells 세스 facilitate rapid transfer between them.

First, place adult hermaphrodites into egg salt solution, which is osmotically balanced 세스 prevent embryos from bursting. Cut the worms in half 세스 release the developing embryos.

Then, identify two-cell stage embryos. These cells are the blastomeres formed by the division of the fertilized ovum. Use the micropipette with the larger opening 세스 transfer an embryo 세스 hypochlorite solution, bleach, which will begin 세스 break down the protective eggshell.

After only 40 세스 55 seconds, transfer the embryo 세스 Shelton’s growth medium, or SGM. Wash each embryo briefly in two wells of SGM 세스 remove all traces of the bleach before placing them in a third well.

Now, use the smaller micropipette 세스 repeatedly pipette the embryo providing the mechanical force 세스 remove the eggshell and expose the blastomeres. Gently continue 세스 pipette the embryo 세스 separate the individual blastomere cells. In the following protocol, we will see this technique in action.

– Hold each end of a microcapillary at a capacity of 10 microliters with right and left hand. Pull the microcapillary towards both ends 세스 apply tension and bring the center of the capillary over a burner 세스 make two hand-pulled capillaries.

Under the dissecting microscope, trim the tips of the hand-pulled the capillaries with forceps. Make the two tip opening sizes approximately two times and one times the short axis length of C. elegans embryos for the embryo transfer and eggshell removal, respectively.

Attach the pulled capillary into a mouth pipetting apparatus. Pipette 45 microliters of egg salt solution onto a well of a multi-well slide.

Place 5 세스 10 adult C. elegans onto the well. To obtain early C. elegans embryos, cut adults into pieces by positioning two needles 세스 the right and left of C. elegans body and sliding the needles past each other.

Pipette 45 microliters of hypochlorite solution onto a well next 세스 the well containing egg salt solution, and pipette 45 microliters of Shelton’s growth medium onto the subsequent three wells. Transfer one-cell stage and early two-cell stage embryos into the hypochorite solution by the mouth pipette with larger size opening and wait for 40 세스 55 seconds.

Wash the embryos by transferring them into the next three wells with Shelton’s growth medium with 1 세스 2 seconds in each of the first two wells. Using the hand-drawn capillary with a smaller size opening, carefully repeat pipetting the embryo for eggshell removal.

After that, continuously pipette with the hand-drawn capillary 세스 separate the two-cell stage embryonic blastomeres.

– After eggshell removal, minimize the force in pipetting embryos up and down 세스 prevent burst.