Isolation of Inner Cell Masses From Mouse Blastocysts by Immunosurgery or Exposure to the Calcium Ionophore A23187

Abstract

Two techniques were evaluated for their use in routinely isolating inner cell masses from mouse blastocysts by destroying the trophectoderm. The most efficient method of immunosurgery was a 15-min incubation in a 1:50 dilution of rabbit anti-mouse spleen antiserum followed by a 30-60 min incubation in guinea pig complement (1:10). Inner cell masses were isolated by incubating blastocysts in 10-5 M Ca ionophore A23187 in medium devoid of Ca and Mg ions. Inner cell masses re-exposed to immunosurgery or the ionophore were less susceptible to lysis than was the trophectoderm. The presence of the zona pellucida reduced trophectoderm lysis by immunosurgery in antiserum dilutions greater than 1:100, but had no effect when in the presence of ionophore. Inner cell masses were consistently isolated from expanded blastocysts which were collected 78 h after ovulation and cultured in vitro for 24 h before ionophore or immunosurgery exposure. Blastocysts which developed for the full 102 h in vivo were frequently unaffected.