Figure 3. Estimation of F-actin accumulation at the NKIS in 3-dimensions.
(A) A 3-dimensonal reconstruction of a series of confocal images collected through the z-axis compressed into a single image of the same ex vivo NK cell (top) and K562 cell (bottom) shown in Figure 1. Fluorescent detection of perforin using FITC-conjugated anti-perforin (light blue) and F-actin using AlexaFluor 568-conjugated phalloidin (red) are demonstrated. The 3-dimensional orientation is shown on the left and an overlay of all fluorescent channels on the right.
(B) Using rectangular cuboids having x,y dimensions of 1µm instead of squares, the same regions defined for 2-dimesnsions in Figure 1 (“A”, “B”, “C”, “D1”,“D2”, and “E2”) were measured in the same 15 cells measured in that figure. Similarly, the accumulation of F-actin at NKIS was estimated as demonstrated in Figure 2 but in 3-dimensons. The horizontal bar in each series of the scatter plot represents the mean and vertical bars represent the SD. Differences between the values obtained for F-actin content in 3-dimensons of “A” vs. “D1”,”C” vs. {A−(B+C)}, “C” vs. (A−D1), and “C” vs. {A−(D2+E2)} were significant, *=p<0.05.