Anoikis-resistant HEK293ar cells survives anoikis through cell-cell contacts. An anoikis-resistant HEK293 cell subpopulation (HEK293ar) was obtained by sequential cycles of adhesion and suspension culture. A. Anoikis in HEK293ar cells in suspension culture at different time points. Flow cytometry (histogram) shows that the apoptosis rate changed little during suspension culture of HEK293ar. The x and y-axes indicate the size of DNA and the number of cells counted, respectively. FL3-H, a standard term for flow cytometry, represents measurement of the fluorescence intensity of propidium iodide (PI) at a super-red wavelength (670 nm). The results are means ± S.D. (n = 4-6). B. The morphology of HEK293ar and parental HEK293 cells in suspension and adhesion culture revealed by microscopy (n = 4-10). Magnification: ×200. C. TEM of a multicellular HEK293ar spheroid. Chromatin masses of moderate electron density are dispersed in the nuclei and nucleoli are conspicuous (arrowed). The cells were observed in 4-6 independent sections. Bar, 2 μm. D. A junctional complex with thickened membranes (arrowed) in suspended HEK293ar cells viewed under SEM. Bar, 50 nm. The cells were viewed in 4-10 independent sections (at least 100 cells/section). E. EDTA (10 mmol/l) treatment disrupted the cell-cell contacts and resulted in massive apoptosis as determined by TUNEL assay. Arrowheads show that single cells detached from the aggregates give more intense TUNEL staining. Magnification: ×200. The apoptotic nuclei were counted in 4-10 independent sections (at least 500 nuclei/section).