Delineating the timing of NK-cell signal collection for activation. (A) Set up. C57BL/6 mice were injected intravenously with PBS or 100 μg poly I:C. After 5 minutes, mice were killed, and LNs were immediately harvested. LNs were dissociated or cultured at the air–liquid interface as a LNOC for 24 hours. (B) NK-cell activation occurs in organ culture but not in cell suspension. NK cells were assessed for expression of CD69 and intracellular granzyme B. Histograms were gated on CD3⁻NK1.1⁺ cells and correspond to LNs obtained from mice treated with poly I:C (black line) or PBS (filled gray). (C) C57BL/6 mice were injected intravenously with PBS or 100 μg poly I:C. After 5 minutes, LNs were placed in organ culture for various periods of time and then dissociated and cultured as a cell suspension so that the total duration of the experiment remained fixed at 24 hours. Cells were analyzed for CD69 up-regulation and intracellular granzyme B content by flow cytometry. (D) CD69 up-regulation was graphed as a function of the duration of the LNOC (mean ± SEM). (E) The percentage of NK cells with high intracellular granzyme B content was graphed as a function of the duration of the LNOC (mean ± SEM). Representative of 3 independent experiments.