3.4.3. Cell-Binding Studies

CD20-expressing Raji cells (humanoid lymphoblast-like B-lymphocyte cells) were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). The cells were cultured in tissue culture flasks (Cellstar; Greiner Bio-One, Kremsmuenster, Austria) using RPMI-1640 medium with 10% (*v*/*v*) fetal bovine serum (FBS) as supplement (Invitrogen Corporation, Lofer, Austria). For cell binding studies 10 <sup>×</sup> <sup>10</sup><sup>6</sup> cells were washed twice with fresh medium, diluted with PBS to a final concentration of 1 <sup>×</sup> <sup>10</sup><sup>6</sup> cells per mL and 500 µL of cell suspension was transferred to Eppendorf tubes. Hereafter, 50 µL of RTX–TCO or non-modified RTX as negative control (both 0.5 µM) was added and the cell suspension was maintained at 37 ◦C under gentle shaking. After 1 h the suspension was centrifuged (2 min, 11 <sup>×</sup> <sup>10</sup><sup>3</sup> rcf), the supernatant was discarded and the cells were washed twice with 600 µL PBS and finally resuspended with 450 µL PBS. Subsequently 50 µL of the radioligand solution (22 nM) was added and the suspension was incubated for 30 min at 37 ◦C. After centrifugation and two washing steps with 600 µL PBS, the cells were resuspended in 500 µL PBS and transferred to polypropylene vials for gamma counter measurement followed by calculation of cell-associated activity in comparison to the total activity applied (*n* = 3, six replicates).
