Fig. 6. Analysis of caspase-mediated cell death in unfixed embryo cell suspensions. (A,B) Representative dual colour cytometry from wild type and Igf2+m/-p embryos at E9.25 (determined by wild-type embryo cell number) labelled with FAM-VAD-FMK and propidium iodide (PI). FAM-VAD-FMK labels cells with active caspases (1-9) and propidium iodide labels cells with disrupted membranes. FAM-VAD-FMK and PI-positive cells are undergoing apoptosis (area iii), PI-positive cells have disrupted membranes and may be in the late stages of cell death (area iv), FAM-VAD-FMK-positive but PI-negative cells maybe in the earlier commitment phase of cell death (ii) and cells with little caspase activity and intact membranes are viable (i). Numbers indicate mean percentage of whole embryos suspension in each quadrant as in C. (C) Summary of mean±s.e.m. for profiles as in A,B for E8.5± 0.25 (n=11), E9.25 (n=22) and E10.5± 0.25 (n=13) embryos. Significant differences occurred around E9.25, with increased caspase activity and cell permeability in cells from Igf2+m/-p embryos (P=0.044).
