Measurement of PIP₃ levels in response to SDF-1 stimulation in Lin^–Sca⁺Kit^– and Lin^–Sca⁺Kit⁺ cells using a selective fluorescence probe. Representative images for unstimulated Lin^–Sca⁺Kit⁺ (control) cells (A-B) and cells stimulated with SDF-1 for 5 minutes (C-D). Cells were incubated with DNM PH GRP1 GFP (A,C) or wild-type PH-GRP1 GFP (B,D). (A-D) GFP staining (i); propidium iodide (nuclear) staining (ii); and (iii) merged GFP/propidium iodide images of X, Y split images shown in (i) and (ii). Original magnification, ×40. (E) Unstimulated cells (NA) and cells stimulated with SDF-1 for 5 minutes were labeled with either a dominant-negative form of PH GRP1 GFP (DMN PH GRP1 GFP; nonspecific binding; ▦) or wild-type PH GRP1 GFP (total binding; ▪). GFP fluorescence is expressed as mean fluorescence intensity and data are presented as the mean plus or minus SEM from 3 experiments; fluorescence measurements were performed on 12 to 15 cells from cytospin preparations prepared in duplicate.