Figure 2.

Absence of Ig secretion in vitro and in vivo by CD19^Cre/+prdm1^flox/Flox B-1 cells. (A) Anti-IgM ELISA performed on supernatants from B-1 cells after enrichment and in vitro culture. B-1 cells were plated at a density of 10⁶ cells/ml for 4 d with no stimulation. Graph represents five control and eight CD19^Cre/+prdm1^Flox/Flox samples. (B) Summary of data from two immunohistochemical experiments for the percentages of cytoplasmic Ig⁺ WT B-1 (filled bars, 33.8 and 29.5%), CD19^Cre/+prdm1^Flox/Flox B-1 (open bars, 9.2 and 10.2%), and 4-d LPS-treated splenic B-2 (gray bars, 39.1 and 37.9%) B cells. Percentages were determined by dividing the fraction of cytoplasmic Ig⁺ cells (WT, 68 Ig⁺/273 total and 114 Ig⁺/558 total; CKO, 23 Ig⁺/332 total and 32 Ig⁺/462 total) by the purity of the respective cultures (WT, 73.8 and 69.3% Mac1⁺IgM⁺; CKO, 74.9 and 67.5% Mac1⁺IgM⁺). The fraction of cytoplasmic Ig⁺ splenocytes (C; 63 Ig⁺/161 total and 109 Ig⁺/297 total) was determined directly because the culture was assumed to be near 100% pure. (C) Bar graph showing data from one representative anti-IgM ELISA experiment for untreated (no bars) or LPS-treated (bars) control (filled bars) and CD19^Cre/+prdm1^Flox/Flox (open bar)-purified B-1 cell cultures. ELISA was performed as in A. (D) Anti-T15 ELISA against serum harvested from 6–10-wk-old CKO (filled squares, n = 8) and control (open squares, n = 6) mice. Filled bars represent the SEM values. Units are OD₄₀₅.