Cell adhesion is impaired by ibrutinib in ibrutinib-sensitive cell lines, but not in ibrutinib-resistant cell lines. (A) Sensitivity of MCL cell lines to ibrutinib according to both cell growth and MTT assay.33 (B) Cell adhesion assay of various MCL cell lines treated with ibrutinib. MCL cells were fluorescently labeled with anti-IgM antibody and allowed to adhere to unlabeled pre-plated NKTert cells in the presence or absence of ibrutinib 400 nM. After 24 hours of culture, plates were washed with saline, and attached and labeled MCL cells were then counted. Data shown represent 1 of the 3 independent experiments. Error bars represent mean ± SEM of 6 replicate reactions. (C) Confocal microscopy images of MCL (green) cells and BMF (red) cells after treatment with DMSO or ibrutinib 400 nM for 24 hours. DAPI staining was added to aid with cell counting. Scale bar, 50 µm. A 3 × 3 montage with z-stack was performed for 4 random fields per treatment. The assay was repeated twice in duplicate. (D) Quantitation of confocal images using the ImageJ cell counter (National Institutes of Health, Bethesda, MD). MCL and BMFs were counterstained with DAPI, and all nuclei were counted. The MCL cells were manually counted by using the cell counter plug-in. The number of BMFs per field was determined by subtracting the number of MCL (green) cells from the total number of nuclei. The ratio of MCL:BMFs was then calculated as a measure of cell adhesion. Error bars represent mean ± SEM. Statistical analysis was done by using an unpaired Student t test. **P < .01; ***P < .0005; ****P < .0001. NS, not significant.