C. elegans Blastomere Dissection: A Method to Remove the Eggshell and Dissociate Embryonic Cells
Trascrizione
– 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 di wells a facilitate rapid transfer between them.
First, place adult hermaphrodites into egg salt solution, which is osmotically balanced a prevent embryos from bursting. Cut the worms di half a 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 a transfer an embryo a hypochlorite solution, bleach, which will begin a break down the protective eggshell.
After only 40 a 55 seconds, transfer the embryo a Shelton’s growth medium, or SGM. Wash each embryo briefly di two wells of SGM a remove all traces of the bleach before placing them di a third well.
Now, use the smaller micropipette a repeatedly pipette the embryo providing the mechanical force a remove the eggshell and expose the blastomeres. Gently continue a pipette the embryo a separate the individual blastomere cells. In the following protocol, we will see this technique di action.
– Hold each end of a microcapillary at a capacity of 10 microliters with right and left hand. Pull the microcapillary towards both ends a apply tension and bring the center of the capillary over a burner a 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 a 10 adult C. elegans onto the well. To obtain early C. elegans embryos, cut adults into pieces by positioning two needles a 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 a 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 a 55 seconds.
Wash the embryos by transferring them into the next three wells with Shelton’s growth medium with 1 a 2 seconds di 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 a separate the two-cell stage embryonic blastomeres.
– After eggshell removal, minimize the force di pipetting embryos up and down a prevent burst.
