Fig 2: CD21 expression on stable DG75 cells driven by CMV promoter is down-regulated over time. DG75 cells were stably transfected with the pcDNA3.1(+)-CD21 plasmid, and 30 d following transfection, CD21+ cells were selected by binding to anti-human CD21 IgG antibody followed by purification with anti-mouse IgG Dynabeads. The purified CD21+ DG75 cells were allowed to grow for 3–5 d and then negative selection was performed to remove the bead-bound cells. The proportion of CD21+ DG75 cells was examined by FACS staining at 7, 14, 34, and 42 d after selection, and is marked on each chart. The black line represents cells stained with anti-CD21 antibody coupled with the secondary fluorescent antibody and the light grey shading shows unstained cells. The experiment was performed in duplicate. The secondary only antibody control for DG75 cells was ignored as it was shown to be no background on DG75 cells.

Fig 3: Constructing a CD21-expressing plasmid under CMV promoter. (A) The human CD21 gene, together with a signal peptide, was cloned into the pcDNA3.1(+) (short as pcDNA) expression vector under a CMV promoter. Picture was modified from the plasmid map of Invitrogen. (B) The generated CD21 expression plasmid pcDNA3.1(+)-CD21(abbreviated as pcDNA-CD21) was confirmed by endonuclease restriction enzyme BamHI, and the uncut pcDNA and pcDNA-CD21 plasmids were used as control, the pcDNA and pcDNA-CD21 were cut at 37°C for 1 h using BamHI, and corresponding predicted bands were 5428 bp for pcDNA and 6596 bp+2001 bp for pcDNA-CD21, which were shown in the gel.

Fig 4: Expression of CD21 on DG75 cells using generated pcDNA3.1(+)-CD21. (A) The generated CD21 expression plasmid (DG75-pcDNA3.1(+)-CD21) was tested by transient expression of 293 cells for 3 d, which was confirmed by immunostaining. LCLs and DG75 cells were used as positive and negative controls for CD21 expression, respectively. Untransfected 293 cells were also compared in parallel as a negative control. Both immunofluorescent and phase-contrast pictures were taken, and scale bars represent 100 μm. (B) Stable transfection of DG75 cells with pcDNA3.1(+)-CD21 plasmid was performed by electroporation. FACS analysis of untransfected cells (DG75) and cells transfected with pcDNA3.1(+)-CD21 plasmid (DG75-pcDNA3.1(+)-CD21) or empty vector (DG75-pcDNA3.1(+)), the expression of CD21 was tested after establishment of the stable cell line, and the selected CD21+ DG75 was confirmed by FACS analysis. The grey shading represents unstained samples alone, the black line represents samples stained with secondary fluorescent antibody alone, and the light dark line represents samples stained with anti-CD21 antibody coupled with secondary fluorescent antibody. pcDNA3.1(+) is shortened to pcDNA for convenience.