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. Author manuscript; available in PMC: 2015 Aug 17.
Published in final edited form as: Mol Ther. 2008 Jan 29;16(3):580–589. doi: 10.1038/sj.mt.6300404

Figure 3. Selection of the Sleeping Beauty-engineered T cells using Rituxan.

Figure 3

(a) PBL. The optimal amount of biotin-Rituxan used for selection of transfected PBL and umbilical cord blood (UCB) T cells was predetermined by staining T cells of PBL (PBL2-19BBζ/CD20) and UCB (UCBCD3+-19BBζ/CD20) expressing 19BBζ/CD20 with a series of biotin-Rituxan antibody dilutions. Two to four weeks after nucleofection, the T cells were harvested, washed once with magnetic cell sorting buffer (Miltenyi), resuspended, and incubated with human FcR blocking reagent (Miltenyi) and biotin-Rituxan (1–2 μg per 106 cells). The cells were washed and mixed with antibiotin microbeads (Miltenyi). After being washed, the cell pellet was resuspended and run through MS or LS columns (Miltenyi). (b) UCB. This experiment was repeated seven times and two times, respectively, with PBL and UCB, yielding similar results. CAR, chimeric antigen receptor; E/T ratio, effector/target ratio; FITC, fluorescein isothiocyanate; Ig, immunoglobulin; SSC, side scatter.