Two distinct NK cell–activating immune synapses. (A-D) NK cells were coincubated with macrophages or macrophages^{high LPS} for 20 minutes, and conjugates were fixed, stained, and imaged. The left panels show the frequency of conjugates where the indicated protein accumulated at the IS, with the number of conjugates imaged indicated above each bar; middle panels, the fold increase of the fluorescence intensity at the synapse relative to elsewhere at the cell membrane, determined from 3D reconstructions of conjugates; right panels, the percent of fluorescence at the IS as a fraction of the total cell fluorescence in the cell, calculated from 3D reconstructions of conjugates. Figures show data for (A) DAP10 and ICAM-1, (B) DAP 10 and NKG2D, (C) DAP 10 and CD3ζ, and (D) DAP 10 and 2B4. In panels A-C, measurements of fluorescence intensities (middle and right panels) were made on synapses with macrophages^{high LPS}, whereas in panel D calculations of 2B4 and DAP10 recruitment (middle and right panels) are made for synapses with (unstimulated) macrophages (Mac.) or macrophages^{low LPS} (Mac.^{low LPS}). For 3D reconstructions, at least 27 synapses were analyzed in each case. (E) NK cells and macrophages^{high LPS} coincubated for different times were fixed and stained for ICAM-1 and DAP10. Reconstructions of the distribution of ICAM-1 (green) and DAP10 (red) at the face of the IS are shown, with the frequency of such distributions marked. Thirty synapses were analyzed for each time point over 3 independent experiments. (F) NK cells were coincubated for 20 minutes with macrophages^{high LPS}, and conjugates were fixed, stained, and imaged. The top row shows the bright field image and associated fluorescence within the boxed area marking NKG2D and DAP10. Reconstructions of the distribution at the face of the IS are shown (lower rows), with the frequency of such distribution marked. For the distribution at the face of the IS, about 30 synapses were analyzed over 3 independent experiments. Scale bars, 10 μm.