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. 2023 Oct 31;11:1270398. doi: 10.3389/fcell.2023.1270398

FIGURE 6.

FIGURE 6

(A,B) Effect of pre-treatment with ibrutinib on CD20 expression levels and obinutuzumab-induced PCD. Raji cells were either left untreated (circles), or pre-treated with ibrutinib for 1 h (squares), and then cultured in the presence of M2-mCherry (triangles) and 10 μg/mL obinutuzumab [all four conditions (B)] for further 24 h. After 24 h, cells were collected and either left unlabelled, or incubated with isotype control (mouse IgG2b-APC), or anti-CD20-APC antibody (0.24 μg/mL) (A), or labelled with 7-AAD and AnnexinV (B) Percentage of positivity to the antibodies was measured as % of mCherry-cells which were APC+. The MFI (geometric mean fluorescence intensity) measured for isotype controls was then subtracted by the MFI of stained samples. Survival percentages were calculated by labelling cells with 7-AAD and AnnexinV. The percentage of cells AnnexinV+ve/7-AAD+ve were calculated and are expressed as a fold change relative to untreated Raji cells alone. Data is the mean + SEM of three independent experiments performed in triplicate.