Sensitivity of Ramos-EHRB clones to anti-Fcμ mAb following transfection with ΔCD79b.
(A) Untreated cells (NT) and 4 ΔCD79b-transfected clones of Ramos-EHRB were cultured (5 × 105/mL) in the presence of anti-Fcμ mAb for 24 hours before assessing apoptosis via flow cytometry using the annexin V–FITC and PI as detailed in Figure 2. Apoptosis was also verified using the hypo-PI method to detect fragmented DNA (data not shown). The histogram shows average results, ± SD, for 3 experiments on each clone. (B) The expression of CD79b and ΔCD79b in the nontransfected (NT) and transfected clones, 4, 9, 12, 23, was confirmed by RT-PCR as detailed in Figure 1. Ratios show the relative level of ΔCD79b/CD79b. GAPDH message was also determined as a control to ensure that equal amounts of DNA were present in each sample. (C) The levels of surface BCR on wild-type and ΔCD79b-transfected Ramos-EHRB clones as measured by flow cytometry. The cells were stained with FITC-conjugated mAb to CD79a (ZL7-4), CD79b (ZL9-1), sIg (M15/8), and an irrelevant control mAb (Irr, CP1/17). No difference was seen in the various clones compared with untransfected cells.
