Fig. 4.

Pore formation in the membrane of OVA-B16 cells induced by tumor-specific CTLs. a–c Splenic CD8⁺ T cells from OT-I mice were sorted by FACS and activated by anti-CD3/CD8 beads for 48 h. Then, the T cells were incubated with OVA-B16 cells at a 20 : 1 ratio for 4 h. The suspended CTLs were removed and the remaining CTLs and OVA-B16 cells were fixed for AFM imaging. AFM topographies are shown (a). The diameters of the long axis or short axis and the depths of the pores were calculated by performing a section analysis of the high-resolution AFM topographies (b). Additionally, the number of pores that formed was counted within three 5 × 5 μm² areas from one cell (n = 6) (c). d OVA-B16 cells were treated with PBS or biotin-labeled SLO (1000 U) and stained with Alexa Fluor 647-conjugated streptavidin. The same cell was imaged using AFM and confocal fluorescence microscopy. Using the topography from AFM and bright field images from confocal microscopy, a merged image of the cell depicting the high-resolution AFM topography and fluorescence is shown. Bars (upper panel), 5 μm; bar (lower panel), 1 μm. e, f OVA-B16 cells were treated with SLO (100 U) for 10 min and imaged using AFM with SLO antibody-conjugated AFM tips. Representative images of OVA-B16 cell topographies and force curves generated from the areas lacking pores and from the border of pores are presented (e). The distribution of the frequency of adhesion force obtained from the data points (n = 712) is shown (b). Asterisks indicate ruptures. White arrows indicate pores