Leukocyte isolation from the maternal peripheral blood and myometrium

Dams mated with B6 CAG-OVA or non-CAG-OVA males were euthanized at 4.5 dpc, 10.5 dpc, 16.5 dpc, 18.5 dpc, and in the postpartum period and peripheral blood was obtained by cardiac puncture. Non-pregnant females were also included as controls. Myometrial tissues from the implantation sites were collected (n = 2 – 14 each), and images of the uterine horns were taken. Isolation of leukocytes from myometrial tissues was performed as previously described [84]. Briefly, tissues were minced using fine scissors and enzymatically digested with StemPro Cell Dissociation Reagent (Life Techologies) for 35 min at 37°C. Leukocyte suspensions were filtered using a 100-μm cell strainer (Fisherbrand; Fisher Scientific, Fair Lawn, NY) and washed with FACS buffer [0.1% BSA and 0.05% sodium azide (Fisher Scientific Chemicals) in 1X PBS] immediately prior to immunophenotyping.

Dams at 10.5 dpc (mid pregnancy) and 16.5 dpc (late pregnancy) were euthanized and peripheral blood was obtained by cardiac puncture (n = 9 – 12 each). Myometrial tissues from the implantation sites were collected (n = 9 – 11 each). Isolation of leukocytes from myometrial tissues was performed, as previously described [84]. Isolated leukocytes were utilized for immunophenotyping.