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. 1997 Mar 17;185(6):993–1004. doi: 10.1084/jem.185.6.993

Figure 2.

Figure 2

Effect of B cell activation on mSLAM expression, and expression of SLAM isoforms in activated B cells. (A) B cells negatively selected by magnetic beads were cultured in the presence of medium only, IL-4 (100 U/ml), anti-CD40 mAbs (10 μg/ml), SAC (0.005%), or anti-μ mAbs (10 μg/ ml) for 6, 20, 48, or 72 h as indicated. Thereafter, the cells were harvested, washed and stained with mAb CD20-FITC and PE-conjugated SLAM-specific mAb A12 or PE-conjugated mouse IgG1 Ab with irrelevant specificity. Open and stippled histograms represent stainings with mAb A12 and control Ab, respectively. PI was always added to exclude dead cells. mSLAM expression on CD20+ cells was analyzed using FACScan® flow cytometer and CellQuest software. A representative of three experiments is shown. (B) B cells purified by cell sorting were cultured in the presence of anti-CD40 mAbs and IL-4 for 24 h, whereas PBMC were cultured in the presence of PMA (1 ng/ml) and Ca2+ ionophore A23187 (500 ng/ml) for 4 h. The cells were harvested and washed, and expression of different isoforms of SLAM was analyzed by RT-PCR using primers specific for each isoform as described in experimental procedures (m, membrane; s, soluble; c, cytoplasmic; vm, variant membrane). HPRT mRNA was amplified as a positive control.