Journal: Molecular and Cellular Biology
Article Title: Negative Regulation of Interleukin-2 and p38 Mitogen-Activated Protein Kinase during T-Cell Activation by the Adaptor ALX
Figure Lengend Snippet: Intact T-cell-dependent B-cell responses to antigen in vivo. (A) Serum immunoglobulin levels from wild-type and ALX-deficient mice were analyzed by ELISA. The data represent averages for six wild-type and nine ALX-deficient mice between 12 and 15 weeks of age. The error bars reflect the standard deviations within each group. (B) Five wild-type and four ALX-deficient mice between 9 and 10 weeks of age were immunized with 100 μg alum-precipitated NP-KLH/mouse on day 0, boosted on day 47, and sacrificed on day 54. Sera were collected at the time points shown and were assayed for the concentration of high affinity anti-NP IgG compared to that of the NP-specific IgG1 monoclonal antibody B1-8 used as a standard. The points represent the average concentrations for all wild-type mice or ALX-deficient mice. Error bars reflect the standard deviations within each genotype. (C) Purified B cells from wild-type and ALX-deficient mice were labeled with CSFE and either were left unstimulated or were stimulated with a combination of anti-IgM, anti-CD40, BLyS, CpG, and/or LPS as shown in the figure and as described in Materials and Methods. After 3 days, the cells were analyzed by FACS. TOPRO-3 (100 nM) was added to exclude dead cells from further analysis. A fixed number of 6-μm beads were added to each sample to permit calculation of the absolute number of live cells within each sample. The y axis represents absolute numbers of live B cells. For each stimulation condition, the gray-filled histograms represent wild-type B cells and the solid black lines represent ALX-deficient B cells. The results shown are representative of three separate experiments. WT, wild type; ALX KO, ALX deficient.
Article Snippet: The following fluorescence-activated cell sorter (FACS) reagents were used: B220-allophycocyanin (APC)-Cy7 (eBiosciences), AA4.1-APC (eBiosciences), CD43-fluorescein isothiocyanate (FITC) (Pharmingen), immunoglobulin M (IgM)-biotin (Southern Biotechnology), streptavidin-peridinin chlorophyll a protein-Cy5.5 (Pharmingen), CD4-phycoerythrin (PE) (Caltag), CD8-APC (Caltag), CD44-FITC (Pharmingen), CD62L-PE (Pharmingen), CD4-biotin (Pharmingen), CD25-APC (Pharmingen), and CD69-FITC (Pharmingen).
Techniques: In Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Purification, Labeling, FACS