PLC-γ2 is required for calcium mobilization, but not for conjugate formation or MTOC reorientation. (A) CFSE-labeled WT and Plcg2^-/- NK cells were coincubated at a 1:1 ratio with PKH-26-labeled YAC-1 target cells for 1, 2, 5, and 10 minutes at 37°C. Percentages of NK cells conjugated with targets were measured by flow cytometry. Data at 5 minutes from one representative experiment of 3 independent experiments is shown. (B) Anti-NK1.1-coated WT NK cells but not Plcg2^-/- NK cells responded to cross-linking secondary antibody with an increase of intracellular calcium. The right panel shows negative control data obtained with NK cells that were left uncoated. (C) WT and Plcg2^-/- NK cells were able to polarize the MTOC and the cytotoxic granules toward CFSE-labeled YAC-1 target cells. The blue staining identifies γ-tubulin (MTOC), whereas the red staining identifies perforin. One representative 2-dimensional reconstruction of the xy projection of a NK/YAC conjugate is represented for both genotypes. Confocal microscopy was carried out using a 63×/1.4 NA oil objective lens. Images were processed using Adobe Photoshop 5.0 (Adobe Systems, San Jose, CA).