Tyrode s acid

For C. elegans embryos, embryonic cells were isolated as described before (Edgar, 1995 (link); Park and Priess, 2003 (link)), with the following modifications. After cutting the adult worms in egg salt buffer, embryos were placed in the freshly made hypochlorite solution [75% Clorox (Clorox) and 2.5 N KOH] for 50 seconds. After washing with Shelton’s growth medium twice (Shelton and Bowerman, 1996 (link)), embryos were put into Shelton’s medium on the coverslip with metallic holds. Eggshell and permeabilization barrier were removed by repeated mouth pipetting with hand-drawn glass microcapillary tubes (10 microliters, Kimble Glass Inc.) to obtain eggshell and permeabilization barrier-free embryos. We call these embryos eggshell-free embryos for simplicity. For blastomere isolation, 2-cell stage eggshell-free embryos were further drawn to separate the two cells. ABx, ABxx, EMS and P₂ cells were isolated by separating daughter cells after each cell division.
Two-cell stage mouse embryos were briefly placed in M2 medium containing Tyrode’s acid (Sigma-Aldrich) to remove the zona-pellucida. They were used as zona-free embryos or further separated by repeated mouth pipetting with hand-drawn glass microcapillary tubes to obtain individual blastomeres.