Fig. 4

Measurement of NK cell cytotoxicity by imaging cytometry. a K562 target cells were stained with calcein AM. After 4 h of incubation with 21-day-expanded NK cells (stimulated with IL-15/18/27), fluorescence images show progressive loss of fluorescence intensity of the K562 cells at various E:T ratios. Representative bright field, calcein, and overlay images showing E:T ratio-dependent target cell killing. Original magnification × 100. The graph represents the percentage of viable or dead target cells. *** P < 0.001 was considered signigicant. b A high power view of the calcein AM assay showing the progress of NK cell killing. Nearly all of the target cells were killed in a 10:1 effector-to-target cell sample, while calcein AM-labeled K562 cells were not killed in the control image. Bright-field and fluorescence overlay images of calcein show K562 cells undergoing apoptotic death following interaction with NK cells. The images were derived from a Zeiss LSM 510 microscope (left). The graph (right) represents cytotoxicity against K562 cells with expanded NK cells on days 7, 14 and 21 over the 4 h of the assay. *P < 0.05, ** P < 0.01, ***P <0.001 compared with day 0. Symbols indicate cytokine treatment groups (n = 3 / group): day 0 (▲), day 7 (▼), day 14 (◆), day 21 (red ○), and K562 cells only (●). c Immunoblot analysis for caspase-8, − 9 and − 3 activation. K562 cells were cocultured with primary NK cells for 4 h. Immunoblotting was performed with antibodies specific for caspase-8, − 9 and − 3 and their cleaved forms. β-actin was used as an internal standard. d Protein bands were quantitated by densitometric analysis. The ratio of the intensity of protein bands relative to that of β-actin was calculated. Bar graph represents the relative expression of cleaved caspase-8, − 9 and − 3 proteins. Experiments were repeated three times with similar results