Regulation of c-Rel by IkBe in Murine Lymphocytes Figure 8. Enhanced survival, increased basal expression of CD40 and increased upregulation of BAFF-R ex vivo in IkBe2/2 splenic B cells. Splenic B cells were isolated by CD19 positive selection from three each of IkBe+/+ and IkBe2/2 mice, plated at 106/ml in 6-well plates without stimulation for 48 hours, and analysed by flow cytometry for (A) viability, as% B220+ cells in the live cell gate (FSc/SSc), (B) median fluorescent intensity (MFI APC) due to CD40 expression on CD40+ live B cells, (C) MFI FITC due to BAFF-R expression on BAFF-R+ live cells. Histograms are for one set of randomly paired IkBe+/+ and IkB2/2 B cells, representative of three for each genotype. In a different experiment, splenic B cells were isolated and plated as above, then stimulated with anti-IgM F(ab’)2 fragment 10 mg/ml and anti-IL-4 50 ng/ml (,250 u/ml) for 72 hours. Splenic B cells were analysed for (D)% viability (FSc/SSc), (E) CD40 expression (MFI PE), and (F) BAFF-R (MFI APC) by flow cytometry. doi:10.1371/journal.pone.0024504.g008 PLoS ONE | www.plosone.org 13 September 2011 | Volume 6 | Issue 9 | e24504